CN109641005A

CN109641005A - Treat Fabry disease

Info

Publication number: CN109641005A
Application number: CN201780039255.0A
Authority: CN
Inventors: 郑伟杰
Original assignee: Academia Sinica
Current assignee: Academia Sinica
Priority date: 2016-06-24
Filing date: 2017-06-14
Publication date: 2019-04-16
Also published as: EP3471736A1; US20190225579A1; TW201800391A; US10995067B2; TWI636039B; WO2017222881A1; EP3471736A4

Abstract

This disclosure is the novel use about a kind of polyhydroxy Pyrrolizidine, can be used to prepare a drug to treat Fabry disease.Accordingly, this disclosure provide it is a kind of to treat suffer from or it is doubtful suffer from Fabry disease individual method.This method includes formula (I) compound, its esters, esters or the solvate that a therapeutically effective amount is administered to the individual,Wherein, R₁It is H or with-COR₂The C optionally replaced_1‑3Amine；R₂It is to have alkyl or alkene that at least the naphthenic base of a substituent group or phenyl optionally replace, wherein the substituent group is to select free halogen, alkyl, group composed by alkylhalide group and alkoxy；It uses improvement, slow down, mitigate and/or prevent symptom relevant to Fabry disease.According to the preferable embodiment of this disclosure, formula (I) compound is the chaperone of a human mutant lysosome alpha-galactosidase A.

Description

Treat Fabry disease

The cross reference of related application

Present application advocates the priority of the interim case 62/354,139 in the U.S. of application June 24 in 2016；Its full text is herein It is included in together, for reference.

Technical field

This disclosure is the field about drug chaperone (pharmacological chaperon)；Especially close In natural polyhydroxy Pyrrolizidine (polyhydroxylated pyrrolidine) derivative and its in treat or prevent method The purposes of cloth Rui Shi disease (Fabry disease).

Background technique

Fabry disease (Fabry disease, FD) is a kind of heredity lysosome metabolic disease, in GLA gene Confirm the missense mutation more than 300 (missense mutation).Endoplasmic reticulum (Endoplasmic reticulum, ER) quality The mutain that management can degrade by the coding generation of those mutated genes；Lack lysosome alpha-galactosidase A (α- Galactosidase A, α-Gal A) activity can make in cell accumulation that there is end α-galactose residue neutral sugar nerve Sheath lipid-glycosphingolipid (globotriaosylceramide, GL-3).The sufferer for suffering from FD generally occurs within chronic forms lesion pain Bitterly, gastrointestinal disturbance, children's acra pseudolymphoma angiokeratoma, Myocardial damage send out the symptom such as myocardial inyaretion early.So far The present, FD are still that the most common lysosome stores up one of disease (lysosomal storage disease, LSD), according to a preliminary estimate often I.e. having 1 suffers from FD in 476,000 to 117,000 life birth newborn, and the data have a possibility that being underestimated.Make us being surprised Strange land, recent research report point out that the ratio that Taiwan male suffers from FD is higher, wherein newborn's Screen test discovery every 1,250 Position individual has 1 to suffer from FD.

Replace treatment (Enzyme replacement using the enzyme that recombinant human α-Gal A (rh- α-Gal A) is carried out Therapy, ERT) it can be used to treat FD, and for most sufferers, ERT can improve or stablize its patient's condition.However, ERT's faces Bed application be still limited by low stability in blood of high price, low convenience, zymoprotein and can not penetration rate of blood brain block Factors such as (blood-brain barrier, BBB).In addition, the recombinant protein of injection stability in neutral pH environment is bad, The organized enzyme correctly folded can be reduced in intravital circulating half-life.Therefore, related fields, which needs one kind, can be used to treat FD Or improve the novel method of rh- α-GalA effect.

Chemical (or drug) chaperone treatment is a kind of emerging strategy for treating LSD, assists dashing forward using small molecule The folding for becoming enzyme uses degradation reaction caused by preventing from being controlled as ER mass, then makes mutant enzyme that can be transferred to lysosome. The wild mycin hydrochloride (1-Deoxygalactonojirimycin, DGJ) of deoxy-galactose is that a kind of couple of α-Gal A has suppression It is sugared (iminosugar) to make active hexa-atomic imines, it is known that can be used as a kind of effective drug chaperone to reply Fa Buruishi Mutation α-Gal A in disease cell and tissue.The III clinical trial phase of recent Migalastat (DGJ) is the results show that chemical companion Companion's albumen can be used as the alternative selection for the treatment of FD.Majority is to treat the Fabry disease sufferer with α-Gal A mutation Chemical chaperone albumen is all DGJ derivative or piperidines (piperidine) type imines sugar.In comparison, related fields relatively lacks benefit Use five yuan of imines sugar researching and analysing as the chemical chaperone albumen for the treatment of FD.Recent high flux screening (about 230,000 kind of chemical combination Object) result of study also do not find to can be used as the potentiality molecule of α-Gal A inhibitor or activator.Lack research report to have delayed to use To treat the development of the novel chemical chaperone albumen of FD.

Such as 2,5- dideoxy -2,5- imido grpup-d- mannitol (2,5-dideoxy-2,5-imido-d- Mannitol, DMDP) etc. natural polyhydroxy Pyrrolizidine be a kind of compound to different glycosidases with inhibitory activity.Citing comes It says, the main inventive people of this disclosure has found a kind of novel chemical chaperone albumen relevant to DMDP, can be used to treat Familial splenic anemia (Gaucher disease), wherein the chemical chaperone albumen can increase lysosome alpha-glucosidase (α- Glucosidase, GCase) activity (Cheng et al., Bioorg.Med.Chem.2013,21,5021).In addition, one Recent research also notes that three kinds of DMDP stereoisomers, includes 2,5- dideoxy -2,5- imido grpup-d- altritol (2,5- Dideoxy-2,5-imido-d-altritol, DIA), the C4- epimer DGDP 2- epimer of DMDP (DGDP be), with And C3- epimer DGDP, to α-Gal (coffee bean) all have good inhibitory activity (Ayers et al., J.Org.Chem.2012,77,7777).

In this disclosure, inventor further researches and develops natural polyhydroxy Pyrrolizidine (such as amido-DMDP or ADMDP) Purposes as molecular chaperone protein of different derivatives and stereoisomers, treat or prevent FD accordingly.

Summary of the invention

This disclosure is based on inventors be surprised to learn that the potentiality that certain ADMDP and its derivative are α-Gal A inhibit Agent, and can be used to the folding of assisted mutagenesis α-Gal A；Accordingly, those compounds can be used as the molecular chaperones egg of mutation α-Gal A It is white, and to prepare the drug that can treat or prevent FD.

Accordingly, this disclosure be about it is a kind of to treat suffer from or it is doubtful suffer from FD individual method.This method Formula (I) compound, its esters, esters or solvate comprising administering a therapeutically effective amount to the individual,

Wherein,

R₁It is H or with-COR₂The C optionally replaced_1-3Amine；And

R₂It is the wherein substituent group to have alkyl or alkene that at least the naphthenic base of a substituent group or phenyl optionally replace It is to select free halogen, alkyl, group composed by alkylhalide group and alkoxy；

It uses improvement, slow down, mitigate and/or prevent symptom relevant to Fabry disease.

According to this disclosure certain embodiments, formula (I) compound can inhibit the activity of α-GAL A.

According to this disclosure other embodiments, formula (I) compound is human lysosomal alpha-galactosidase A (α- Galactosidase A, α-Gal A) mutant chaperone.

According to the preferable embodiment of this disclosure, which includes a mutation, selected from by Group composed by R112H, P205T, Q279E, R301Q, R356W and R363C.

According to the preferable embodiment of this disclosure, formula (I) compound be selected from by

Composed group.

According to one better embodiment of this disclosure, formula (I) compound is

According to this disclosure embodiment, formula (I) compound is to be administered with daily 0.001-500 grams of dosage to this In body body；It is 0.05-450 grams preferably about daily.

According to certain preferable embodiments, this method is further included prior to, concurrently with, or after administering formula (I) compound, Mankind α-Gal the A of a therapeutically effective amount is administered to the individual.

Another aspect of this disclosure is the chemistry knot about a kind of novel ADMDP derivative, with formula (I-1) Structure,

Wherein,

R₃It is H or-COR₂；And

R₂It is the wherein substituent group to have alkyl or alkene that at least the naphthenic base of a substituent group or phenyl optionally replace It is to select free halogen, alkyl, group composed by alkylhalide group and alkoxy.

According to a better embodiment, formula (I-1) compound is

According to another better embodiment, formula (I-1) compound is

One or more embodiments of this disclosure can be found in subsequent explanation.By this disclosure detailed description and Scope of the claims can deduce the features of the present invention and advantage.

After refering to following description, the technical field of the invention those of ordinary skill, which works as, can will readily appreciate that the present invention Essence spirit and other goals of the invention and the technology used in the present invention means and state sample implementation.

Detailed description of the invention

For above-mentioned and other purposes, feature, advantage and embodiment of the invention can be clearer and more comprehensible, institute's accompanying drawings are said It is bright as follows:

Fig. 1 is the histogram illustrated according to one embodiment of this disclosure, is about compound 9 to 24 in pH When 4.6 or pH 7.0, to inhibition effect of α-Gal A；

Fig. 2 is the linear graph that is illustrated according to one embodiment of this disclosure, is about (a) compound 17 and (b) Companion's effect of compound 33 or 37 couples of Human Lymphocytes strain N215S；

Fig. 3 is the histogram that is illustrated according to one embodiment of this disclosure, is about (a) compound 17 and (b) The companion's effect of compound 33 to rh- α-Gal A mutant；

Fig. 4 is the linear graph illustrated according to one embodiment of this disclosure, is about compound 17 to rh α-Gal The effect of A fusion temperature；

Fig. 5 is the histogram illustrated according to one embodiment of this disclosure, is about to Fabry disease sufferer Cell strain N125S (a) merges administering compound 17 and α-Gal A, or (b) individually administers effect caused by compound 17；With And

Fig. 6 is the linear graph that is illustrated according to one embodiment of this disclosure, is about (a) compound 33 and (b) Durability of the DGJ to companion's effect of N125S.

Specific embodiment

In order to keep the narration of this disclosure more detailed with it is complete, below for state sample implementation of the invention and specific Embodiment proposes illustrative description；But this not implements or uses the unique forms of the specific embodiment of the invention.Embodiment party The feature of multiple specific embodiments is covered in formula and to construction and the method and step for operating these specific embodiments and its Sequentially.However, can also reach identical or impartial function and sequence of steps using other specific embodiments.

1. definition

Unless otherwise stated, otherwise " amine " (amine) word refers to 1 to 20 (such as 1 to 10,1 to 9,1 To 8,1 to 7,1 to 6,1 to 5,1 to 4,1 to 3,1 to 2 or 1) carbon atom and wrap an at least amido The hydrocarbon of straight chain type, branched chain type and/or ring-type (naphthenic base).Preferably, the amine of this disclosure refers to low-alkylamine, Such as C_1-3Amine.Amine is suitable for the invention as level-one or secondary amine, and unless otherwise stated, it otherwise can be with one or more substituent groups An at least hydrogen atom in individually optional substituted amido, i.e. amine can be the amine (one " unsubstituted amine ") of unsubstituted or be taken with one or more The amine (one " substituted amine ") replaced for base.In some embodiments, amine is benzylidene amino (methylene amine).? It is the hydrogen atom come in the amido of substituted methylene amine with an acyl group in other embodiments.

In a particular implementation, " can be substituted " (substituted) word is describing a chemical structure or official When energy base, refer to that the structure or the derivative of functional group, one or more hydrogen atoms can be with acyl group, alkoxy, alkyl, aryl, halogen Element, alkylhalide group or hydroxyl are replaced.

" solvate " (solvate) word this disclosure refer to by a compound (such as formula of the invention (I) change Close object) with surrounding solvent molecule (such as water, alcohols and other polar organic solvents) formed compound.Alcohols includes first Alcohol, ethyl alcohol, normal propyl alcohol, isopropanol, n-butanol, isobutanol and tert-butyl alcohol alcohols also may include polymerism alcohols, such as poly- second two The polyolefin diols such as alcohol (polyethylene glycol) and polypropylene glycol (polypropylene glycol) (polyalkylene glycol).It is most known from and preferably solvent is water, the solvent that will generally be formed with aqueous solventization Chelate compound is known as hydrate (hydrates).

Unless otherwise stated, otherwise " therapeutically effective amount " (therapeutically effective of a compound Amount) refer to and treating or handling disease or when symptom, it is sufficient to generate treatment benefit, or delay or reduce one or more with The dosage of the disease or the related symptom of symptom.The therapeutically effective amount of one compound refers to the dosage of a healing potion, can be single It solely administers or is administered jointly with other treatment to individual in vivo, use the treatment benefit for generating treatment or handling disease or symptom." Effective quantity " (effective amount) word include can improve wholistic therapy, be reduced or avoided disease or symptom symptom or The origin cause of formation, or increase the dosage of the therapeutic efficiency of another healing potion.

" prevention effective dose " (the prophylactically effective amount) of one compound refers to a compound It is enough to prevent, delay generation or reduce the dosage of disease or symptom occurrence risk in vivo in individual.

" sufferer " (patient) and " individual " (subject) is interchangeable vocabulary in this disclosure, and can be dynamic for one Object or a human individual." FD sufferer " (FD patient) refers to the individual for suffering from FD after diagnosing in this disclosure, and has The mutant form of α-GalA, the mutation can make enzyme that can not form stable configuration in endoplasmic reticulum.Can not be formed structural stability into And will lead to a large amount of α-Gal A and move towards degradation reaction, without being communicated to lysosome.Illustrative α-Gal A relevant to FD Mutation include, but are not limited to, L32P, N34S, T41I, M51K, E59K, E66Q, I91T, A97V, R100K, R112C, R112H, F113L、T141L、A143T、G144V、A156V、L166V、D170V、C172Y、G183D、P205T、Y207C、Y207S、 N215S、A228P、S235C、D244N、P259R、N263S、N264A、G272S、S276G、Q279E、Q279K、Q279H、 M284T、W287C、I289F、M296I、M296V、L300P、R301Q、V316E、N320Y、G325D、G328A、R342Q、 R356W, E358A, E358K, R363C, R363H and P409A.According to the preferable embodiment of this disclosure, unstable α- GalA includes any mutation of R112H, P205T, Q279E, R301Q, R356W or R363C.

Unless otherwise stated, it otherwise " treats " (treat, treating or treatment) word and refers to a movement, when one When individual particular ailment or symptom, which can reduce the severity of the disease or symptom, one or more symptom, or Delay or reduce the process of the disease or symptom.

It should be clear that when the spatial chemistry construction for the part for especially not indicating a structure or a structure with thick line or broken string, The part of the structure or the structure should be read as including its all stereoisomers.Similarly, there are one or more hands in narration When the compound at property center, if the spatial chemistry at those not specified centers, it should be read as including pure alloisomerism Object and its mixture.In addition, in schema any atom with unsaturated valence mumber it will be assumed that its be in conjunction with enough hydrogen atoms, To meet its valence mumber.

Although the numberical range and parameter to define wider range of the present invention are all rough numerical value, herein as far as possible The correlation values in specific embodiment are accurately presented.However, any numerical value is substantially inevitably containing because of individual tests Standard deviation caused by method.Here, " about " typically refer to actual numerical value a certain number value or range positive and negative 10%, 5%, within 1% or 0.5%.Either, " about " word represents actual numerical value and falls within the acceptable standard error of average value, Depending on depending on the persons of ordinary skill in the technical field of the present invention the considerations of.Other than experimental example, or unless otherwise specific Illustrate, when being appreciated that all ranges used herein, quantity, numerical value and percentage are (such as long to describe material utilization amount, time Short, temperature, operating condition, quantitative proportion and other similar persons) by the modification of " about ".Therefore, it is said unless otherwise opposite Bright, this specification and the revealed numerical parameter of subsidiary claim are all rough numerical value, and visual demand and change. These numerical parameters should be at least interpreted as to pointed number of significant digit and apply the obtained numerical value of general transfer method.Herein Place, numberical range is expressed as by end point to another section of point or between two endpoints；Unless otherwise indicated, described herein Numberical range all includes endpoint.

Singular noun used in this specification covers the complex number type of the noun；And also cover the name when used plural noun The singular type of word.

2. the compounds of this invention

The compound of the present invention is the stereoisomers of 1- amido deoxidation-DMDP (1-aminodeoxy-DMDP, ADMDP) And its derivative.The chemical structure of ADMDP includes the asymmetric carbon atom of at least four (i.e. chiral centre), therefore ADMDP includes At least 16 kinds of stereoisomers.The present inventor has developed a kind of strategy, can synthesize according to this disclosure embodiment 1 16 kinds of ADMDP stereoisomers (i.e. compound 9 to 24) and its derivative (i.e. 28,29 and 33-37 of compound).Then to obtaining ADMDP and/or its derivative (i.e. compound 9-24,28,29 and 33-37) carry out various analyses, to assess it as treating The application of the molecular chaperone protein of Fabry disease.Those analysis methods, which include at least, to be inhibited α-Gal A, stablizes recombinant alpha- The folding of Gal A, assisted mutagenesis α-Gal A, and administered jointly when with α-Gal A from the lymphocyte when institute for being originated from FD sufferer Adding for generating multiplies effect.According to this disclosure as a result, the above compound (i.e. formula (I) compound) for stating appraisal procedure confirmation It can be used as a kind of potential lead compound of tool, prepare the therapeutic agent of FD accordingly.

Therefore, the present invention includes formula (I) compound, its esters, esters or solvate,

Wherein,

R₁It is H or with-COR₂The C optionally replaced_1-3Amine；And

R is the wherein substituent group to have alkyl or alkene that at least the naphthenic base of a substituent group or phenyl optionally replace It is to select free halogen, alkyl, group composed by alkylhalide group and alkoxy.

Illustrative formula (I) compound include, but are not limited to,

According to certain embodiments, the compound of this disclosure has the chemical structure of formula (I-1),

Wherein,

R₃It is H or-COR₂；And

In a preferred embodiment, formula (I-1) compound is

In a further preferred embodiment, formula (I-1) compound is

According to this disclosure other embodiments, formula (I) compound is human lysosomal alpha-galactosidase A (α- Galactosidase A, α-Gal A) mutant chaperone.Specifically, formula (I) compound can assist unstable α- The folding of Gal A or the α-Gal A of mutation, wherein α-Gal the A of the mutation include R112H, P205T, Q279E, R301Q, Any mutation of R356W or R363C.

3. application method

The present invention therefore include it is a kind of to treat or prevent one suffer from or it is doubtful suffer from FD individual method.This method Comprising treating or preventing a effective amount of formula (I) compound, its esters, esters or solvate to individual administering one, uses and change It is apt to, slows down, mitigates and/or prevents the symptom of FD.In some embodiments, this method is further included in administering formula (I) compound Prior to, concurrently with, or after, the mankind α-GalA of a therapeutically effective amount is administered to the individual.

According to this disclosure embodiment, formula (I) compound be selected from by

Composed group.

Preferably, formula (I) compound is compound 17,33 or 37, its esters, esters and/or solvate.More preferably, Formula (I) compound is compound 17.Again more preferably, formula (I) compound is compound 33.

According to this disclosure embodiment, this disclosure formula (I) compound is with daily 0.001-500 grams of dosage Administering is internal to the individual, for example, daily 0.001,0.01,0.1,1,10,20,30,40,50,60,70,80,90,100,110, 120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、 310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490 and 500 grams；Preferably, daily about 0.05-450 grams, such as daily 0.05,0.5,1,5,10,20,30,40,50,60,70,80, 90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、 280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440 and 450 grams；More Goodly, daily about 5-400 grams, for example, daily 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,110, 120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、 310,320,330,340,350,360,370,380,390 and 400 grams.

Administering dosage, path and the order of administration of formula (I) compound can be adjusted according to specific factor, such as be intended to treat, The specific symptom of prevention or treatment, the age, gender and sufferer physiology condition shape.Influence of those factors to disease is institute of the present invention Belong to known to technical field those of ordinary skill, and can usually test to analyze and learn.

Formula (I) compound can prepare accordingly medicament (such as pharmaceutical compositions in conjunction with pharmaceutical excipient appropriate or carrier Or pharmaceutical formulations), and administered with paths such as oral, non-oral, percutaneous, part and mucous membranes to FD patient.

The weight of formula (I) compound can account for 0.1% to the 99% of medicament total weight.In some embodiments, formula (I) is changed The weight for closing object at least accounts for the 1% of medicament total weight.In some embodiments, the weight of formula (I) compound at least accounts for medicament The 5% of total weight.In other embodiments, the weight of formula (I) compound at least accounts for the 10% of medicament total weight.Other In embodiment, the weight of formula (I) compound at least accounts for the 25% of medicament total weight.

Certain medicaments are to be formulated as being suitable for oral, mucous membrane (such as in nasal cavity, sublingual, intravaginal, cheek or in rectum), non- It oral (such as subcutaneous, intravenous, the full dosage injection (bolus injection) of single, intramuscular or intra-arterial) or percutaneously throws The single formulation given.Illustrative dosage form include, but are not limited to, tablet, caplet, capsule (such as soft elastic gelatin capsule), Cachet, ingot piece, dispersion, suppository, ointment, poultice, emulsion, emplastrum, solution, patch, spray, gel, is fitted at pastille It administers to the intracorporal liquid dosage form of sufferer to oral or mucous membrane mode (comprising suspension (such as the liquid compatible or insoluble with water Liquid suspension, oil in water emulsion or water-in-oil emulsion), solution and elixir), be applicable in and administered in a manner of non-oral to patient Interior liquid dosage form and sterile solid (such as crystalloid or amorphous solid), can remake for be applicable in administered in a manner of non-oral to The intracorporal liquid dosage form of sufferer.

Medicament of the present invention can be prepared according to specific administering path.For example, oral administration needs enteric coating to coat, Protection the compounds of this invention is used to avoid decomposing in gastrointestinal tract.Similarly, medicament may include additional ingredient, and using will be active Ingredient is transmitted to active position.For example, compound can be administered with micro- rouge body dosage form, to prevent it from being divided by enzyme effect Solution, auxiliary is carried through the circulatory system of individual and penetrating cell film is sent to intracellular locations.

Similarly, the poor compound of dissolubility can be co-formulated as liquid with cosolvent, emulsifier and surfactant Dosage form (and dosage form suitable for recasting), those cosolvents, emulsifier and surfactant include, but are not limited to, cyclodextrin (example Such as alpha-cyclodextrin and beta-cyclodextrin) and anhydrous solvent, it includes, but be not limited to, alcohol, isopropanol, ethyl carbonate, acetic acid Ethyl ester, benzyl alcohol, Ergol, 1,3-BDO, dimethylformamide, dimethyl sulfoxide (dimethyl sulfoxide, DMSO), bio-compatibility oily (such as cottonseed oil, peanut oil, corn oil, wheat-germ oil, castor oil, olive oil, sesame oil), Glycerol, tetrahydrofuran, polyethylene glycol, sorbitol anhydride fatty acid ester and combinations thereof (such as DMSO: corn oil).

Composition, shape and the type of medicament can be adjusted according to specific use demand.For example, compared to a disease Chronic treatment, to same disease carry out acute treatment when, drug may include one or more larger amount of active constituents.It is similar Ground, compared to medicinal preparation for oral administration, non-oral medicament may include one or more less amount of active constituents when treating same disease.This Technical field that the present invention belongs to those of ordinary skill can be appreciated that the otherness between particular agent of the present invention.Such as it can refer to Remin.gton ' s Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990)。

3.1 are suitable for the medicament of oral administration

Medicament comprising formula (I) compound of the present invention can be formulated as being suitable for oral discontinuous dosage form, such as tablet (such as chewable tablets), pastille, capsule and liquid (such as syrup).Those dosage forms include the active constituent of specific quantity, and can foundation It is prepared by method known to person skilled in the art.Such as it can refer to Remington ' s Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

In general, technology can be made according to traditional medicine compound, active constituent is mixed with an at least excipient, according to To prepare peroral dosage form.The excipient of inhomogeneity shape can be used according to specific administering demand.

Based on the characteristic for being easy to administer, tablet and capsule are two kinds of most common peroral dosage forms.If necessary, can be with standard Aqueous or nonaqueous techniques coat tablet.Those dosage forms can be prepared in ethnopharmacology method.In general, can by activity at Point with liquid-carrier, subdivision solid carrier or combinations thereof after evenly mixing, be made as specific shape.It can be to be added in solid dosage forms Disintegrating agent, to assist fast solutions.Also lubricant can be added to assist preparation dosage form (such as tablet).

3.2 are suitable for the medicament of non-oral administering

Non-oral medicament can the modes such as subcutaneous, intravenous (including the full dosage injection of single), intramuscular and intra-arterial throw It gives to patient.Sufferer can be avoided to the defence mechanism of pollutant based on this kind of administering mode, therefore non-oral dosage forms It is generally sterile, or aseptic process is carried out before administering.Illustrative non-oral medicament include, but are not limited to, direct injection Solution, the desciccate that may be dissolved or suspended in the pharmaceutically acceptable carrier to inject, direct injection suspension and Emulsifier.

Carrier suitable for preparing the non-oral medicament of the present invention is ripe for the technical field of the invention those of ordinary skill The carrier known.Illustrative carrier include, but are not limited to, water, liquid-carrier (such as, but not limited to, sodium chloride solution, Lin Ge Family name's liquid and glucose), carrier miscible with water (such as, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol) and non-aqueous load Body (such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate (isopropyl Myristate) and Ergol (benzyl benzoate)).

3.3 are suitable for the medicament of percutaneous, part and/or mucous membrane administering

Percutaneously, portion, area and mucous membrane medicament include, but are not limited to, and ophthalmic solution, mist agent, emulsion, lotion, ointment, coagulates at spray Glue, solution, emulsifier, suspension and other present invention form known to ripe field those of ordinary skill.Such as it can refer to Remington ' s Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990).Percutaneous medicament includes " drug storage type " (reservoir type) or " matrix type " (matrix type) patch, can be in One specific time of skin attachment, uses and the active constituent of given dose is promoted to penetrate skin.

Excipient (such as carrier and diluent) appropriate and other can prepare percutaneous, part and the material of mucosal dosage forms is To substance known to pharmaceutical field those familiar, and can be selected according to the specific organization and its dosage form of pharmaceutical compositions to be administered With suitable excipient.

According to treatment tissue difference, can be given prior to, concurrently with, or after administering inventive compound it is additional at Point.For example, it penetration enhancer can be used to carry out Supplementary active ingredients and be sent to tissue.

Also the pH value of drug or dosage form, or the pH value of administering drug or the tissue of dosage form be can adjust, it is one or more to improve Transmission effect of kind active constituent.Similarly, polarity, ionic strength and the tension of solvent carrier be can adjust to improve transmission function Effect.The compounds such as stearate can be made an addition in drug and dosage form, to change hydrophily or the parent of one or more active constituents Oiliness uses improvement transmission effect.At this point, stearate can be used as the lipid carrier, a kind of emulsifier or surface of a kind of preparation Activating agent and a kind of transmission dose or penetration enhancer.Can using the salt, hydrate or solvate of active constituent come Further adjust the characteristic of composition.

Multiple experimental examples are presented below to illustrate certain aspects of the invention, with sharp common in the technical field of the invention The technical staff implementation present invention, and these experimental examples should not be considered as limitation of the scope of the invention.It is believed that being familiar with this field Personnel can completely utilize in the case of being not required to excessively interpret after having read description proposed herein and practice the present invention.This All open source literatures of place reference, full text are all considered as a part of this specification.

Embodiment

Material and method

Cell culture

By Human Lymphocytes (08C0058) and it is originated from the lymphocyte culture of sick (GM04391) sufferer of Fabry disease In including 15%FBS (fetal calf serum, Invitrogen), 2mML- bran propylhomoserin and 1% penicillin/streptomycin (Gibco) In RPMI-1640 (Gibco) cell culture fluid.By COS-7 cell culture in including 10% thermally treated FBS, 4mM L- bran DMEM (Dulbecco ' s modified Eagle ' the s medium of propylhomoserin and 1% penicillin/streptomycin；Gibco) cell is trained In nutrient solution.By all cell culture in 37 DEG C, include 5%CO₂Moist cell incubator in, culture cell to 1-30 generation When, carry out subsequent bio assessment.

Cell toxicity test is carried out using Human Lymphocytes

By wild type human quasi-lymphocyte (08C0058) plantation in 96 hole disks, cell density is every hole 50,000 A cell.After 24 hours, cell culture fluid is replaced, and the test compound that ultimate density is 10-200 μM is added.All changes It closes object and is all dissolved in DMSO or H₂In O, experimental comparison group is then carried out with DMSO.By cell culture in 37 DEG C of 5%CO₂Middle 48-72 Hour.Later, 10 microlitres of alamarBlue is added to the cell being suspended in cell culture fluid, then the mixture is incubated at 37 DEG C of 5%CO₂It is 3-5 hours total.Survival is quantified and measured with 560 nanometers of excitation spectrum and 590 nanometers of diverging spectrum The quantity of cell.

The in vitro stability of α-GalA

Small molecule is detected in stable alpha-Gal A using Fa Burui enzyme (Fabrazyme, a kind of rh- α-Gal A), is avoided It carries out the effect of degradation reaction.Enzyme (20 microlitres, pH 7.0) and 0,0.5,5 or 50 μM of compound 17-19 are anti-in on ice It answers 10 minutes.A specimen is heated to 48 DEG C (as functions of time), with thermally deactivating (degradation) α-Gal A, is later released a specimen In the 0.1M citrate phosphate buffer (pH 4.6) of dilute to 3 times volumes.Immediately by enzyme-to-substrate (the 4- methyl umbrella of 0.2mM Type ketone α-D- galactoside (4-methyllumbelliferyl α-D-galactoside)) it is reacted 15 minutes in 37 DEG C, then with Glycine buffer extinguishing (quenching) reaction.(excitation spectrum is 355 nanometers to the methyl umbellate form ketone of measurement dissociation, diverging light Spectrum is 460 nanometers).To indicate enzymatic activity relative to the enzyme not heated.

The intracellular reactive of N215S sufferer lymphocyte enhances test

The lymphocyte (GM04391) of Fabry disease N215S sufferer is planted in the 96 hole disks at sterile transparent bottom, Cell density is 20,000 cell of every hole, later by cell culture in 37 DEG C of 5%CO₂Environment 12-24 hours.It connects , by cell culture in environment 4 days comprising or not comprising potentiality compound.Carry out enzyme test: after PBS washing cell 3 times, It is homogenized cell using the triton X 100 for being dissolved in 4.6 buffer of pH (citric acid phosphorus hydrochlorate) of 50 microlitres, 0.1%, with After 14,000g revolving speed centrifugation, 20 microlitres of supernatant and 20 microlitres of substrate solution (the 4- methyl umbellate form ketone comprising 8mM are taken Half lactam of N- acetyl of α-d- galactoside (4-MU- α-Gal) and 150mM are sugared (N-acetylgalactosamine), to press down α-Gal B (N- acetyl gala ammonia carbohydrase (N-acetylgalactosamidase)) processed, is dissolved in 0.1M citrate-phosphate buffer Liquid (pH4.6)) to carry out enzyme test) reacted 1 hour in 37 DEG C.It is added later and stops solution (0.4 mole every liter of K₂CO₃, pH 10.6), using Victor disk reader with 355 nanometers of excitation spectrum and 460 nanometers of diverging spectral measurement fluorescent value.It will be glimmering Light value subtracts background value, to calculate the fluorescence volume only obtained by substrate solution.It is determined using MicroBCA Protein assay set group Protein concentration.Fluorescence values are converted to using every liter of 0 micromole to every liter of 200 micromolar 4- methyl umbellate form ketone standard curves The absolute activity of α-Gal A, and it is denoted as Nai Moer (the nmol/mg protein per of every milligram of albumen per hour h).It with untreated enzymatic activity standardized fluorescent numerical value, uses to obtain the relative activity of α-Gal A, and is indicated with multiple.

Lysosome α-Gal A is grown in choosing

The plastid of full-length cDNA comprising α-Gal A is obtained by RT-PCT.With KOD Hot Start archaeal dna polymerase and just To primer 5 '-AGGTCGGATCCGACAATGCAGCTGAGGAACC-3 ' (SEQ ID NO:1) and 5 '-GGTGGAA of reverse primer Wild type human's GLA gene that TTCTTAAAGTAAGTCTTTTAATGACATCTGCA-3 ' (SEQ ID NO:2) amplification choosing is grown, And import specific limiting enzyme point BamHI and EcoRI.Size is observed as the PCR product of 1.4kb with electrophoretic analysis, and with limitation Enzyme is confirmed.Carrier pJET2.1 is grown into the choosing of amplified production insertion mammal, prepares the carrier construction of mutation accordingly.

The mutation of lysosome α-Gal A

It is prominent with α-Gal A to prepare to pinpoint PCR mutation using QuikChange Lightining rite-directed mutagenesis set group Carrier is grown in the choosing of change.An other nucleotide subsitution is carried out with pcr amplification reaction and QuikChange Lightning enzyme, wherein being Using the pJET2.1/GLA carrier comprising wild type sequence as template, the primer that table 1 lists the 30-40 unit used (has specific The underlying stock of sequence modification and anti-stock primer).Confirm that each Mutant Cytoplasm, Fig. 2 elaborate Taiwan with restriction enzyme and DNA electrophoretic analysis The sequencing result of genome.After cutting pJET2.1/GLA with restriction enzyme BamHI and EcoRI, purified using electrophoretic analysis.

After GLA gene is connected to carrier pcDNA3.1, turn shape into Escherichia coli, uses amplification and generate comprising wild type And the pcDNA3.1/GLA plastid of mutant nucleotide sequence, for the use of subsequent transfection experiment.

Primer of the table 1. to pinpoint PCR mutation

Of short duration transfection COS-7 and chaperone test

By COS7 cell seeding in 24 hole disks, cell density is every hole 1x10⁵A cell is cultivated 16-20 hours Afterwards, the performance plastid of α-Gal cDNA is temporarily transfected comprising human wild type or is mutated using Mirus ILT-1 reagent.Training After supporting 5-8 hours, addition includes or the fresh cell medium of the compound not comprising companion's effect to be tested, culture are 60 small When.Later, cell is collected with 0.1%EDTA- trypsase, and pH4.6 buffer (lemon is dissolved in 100 microlitres, 0.1% Acid phosphoric acid salt) tritonX100 homogenize cell, be then centrifuged with the revolving speed of 14,000g.Detect cellular breakdown products The activity of middle α-Gal A.In simple terms, 10 microlitres of supernatant and 10 microlitres of substrate solution (the 4- methyl umbrella comprising 8mM are taken The N- acetyl gala ammonia of type ketone α-d- galactoside (4-MU- α-Gal) and 150mM are sugared (N-acetylgalactosamine), use To inhibit α-Gal B, (N- acetyl gala ammonia carbohydrase (N-acetylgalactosamidase) is dissolved in 0.1M citric acid phosphorus hydrochlorate Buffer (pH 4.6)) to carry out enzyme test), it is reacted 1 hour in 37 DEG C.The solution of addition stopping later be (0.4 mole every liter K₂CO₃, pH 10.8), it is glimmering with the diverging spectral measurement of 355 nanometers of excitation spectrum and 460 nanometers using Victor disk reader Light value.Fluorescent value is subtracted into background value, to calculate the fluorescence volume only obtained by substrate solution.Utilize MicroBCA Protein assay set Group determines protein concentration.Using every liter of 0 micromole to every liter of 200 micromolar 4- methyl umbellate form ketone standard curves by fluorescence number Value is converted to the absolute activity of α-GalA, and is denoted as Nai Moer (the hmol/mg protein of every milligram of albumen per hour per h)。

Glycosidase activity test

Using multiple detecting reader (SpectraMax M5, Molecular Device) with 405 nanometers of absorption spectrum Or with 355 nanometers of excitation spectrum and 460 nanometers of diverging spectrum come detect various concentration to nitre phenyl-pyrane glucoside (p-nitrophenyl-glycopyranoside) or 4- methyl umbellate form ketone-glucopyranoside (4- Methylumbelliferyl-glycopyranoside) in room temperature or 37 DEG C of hydrolysis initial velocity.It will by GraphPad Data substitute into Michaelis-Menten formula, use and determine Km value and Vmax value.Utilize the commercial source of every milliliter of 0.1 unit Enzyme, 20nMCerezyme (the recombinant human acid of (recombinant human acid alpha-D-glucosidase), 10nM Property alpha-glucosidase) and every milliliter of 62 microgram the α-Gal A from mankind's normal lymphocytes establish it is ideal increase it is bent Line, the test concentrations of inhibitor are then 100 μM.It is tested with 96 hole micro-plates, wherein administering alpha-glucosidase (fat Bacillus alcalophilus (Bacillus stearothermophilus)) and α-Gal (green coffee bean) handle when, be using phosphoric acid Sodium buffer (1O0mM, pH 6.8)；It is administeringAnd beta-glucosidase (almond) When, it is using sodium citrate buffer solution (100mM, pH 5.2)；And in administering alpha-Mannosidase (α-mannosidase, sword bean (Jack bean)) and when α-Gal from mankind's normal lymphocytes, then be using buffered sodium citrate (100mM, pH4.5)。

Thermostabilization Transfer Experiment (Thermal stability shift assay)

Using improved fluorescence heat stabilization test with Rotor-Gene system in medium pH buffer (potassium phosphate, pH 7.0) or acidic pH buffer liquid (citrate phosphate buffer, pH4.6) detects the stability of α-Gal A.In simple terms, will α-Gal A (2 microgram) is uniformly mixed with 17,28 and the 29 of SYPRO tangerine stain and various concentration, and end reaction volume is 20 micro- It rises.Reaction tray is heated with 1 DEG C of temperature gradient per minute, and continues to monitor the fluorescent value of SYPRO tangerine.It degrades in complete temperature Afterwards, the fluorescence intensity of each temperature is standardized with maximum fluorescence value.

α-GalA activity test analysis is carried out after administering Fa Burui enzyme and compound 17 jointly to sufferer lymphocyte

The lymphocyte of Fabry disease sufferer is planted in 96 sterile, transparent hole disks, cell density is every hole 20,000, hole cell, and in 37 DEG C, 5%CO₂Environment in cultivate 12-16 hour.Then method cloth is individually administered to cell Auspicious enzyme (every liter of 1 nanomole), or merge administering Fa Burui enzyme (every liter of 1 nanomole) and compound 17 (every liter 1,10,50 micro- is rubbed You), it reacts 24 hours.With grown cultures liquid washing cell 3 times, later with grown cultures liquid in 37 DEG C, 5%CO₂Environment training It supports 3 days.It carries out enzyme test: after with PBS washing 2 times, 4.6 buffer (citric acid phosphoric acid of pH being dissolved in 50 microlitres, 0.1% Salt) triton X 100 homogenize cell, then with the revolving speed centrifugation of 14,000g after, take out 20 microlitres of supernatant and 20 Microlitre substrate solution (the N- acetyl of 4- methyl umbellate form ketone α-d- galactoside (4-MU- α-Gal) and 150mM comprising 8mM half Newborn ammonia sugar, is dissolved in 0.1M citrate phosphate buffer (pH 4.6)) to carry out enzyme test) reacted 1 hour in 37 DEG C.Later plus Enter to stop solution (0.4 mole every liter of K₂CO₃, pH 10.8), using Victor disk reader with 355 nanometers of excitation spectrum and 460 nanometers of diverging spectral measurement fluorescent value.Fluorescent value is subtracted into background value, to calculate the fluorescence only obtained by substrate solution Amount.Protein concentration is determined using MicroBCA Protein assay set group.Utilize every liter of 0 micromole to every liter of 200 micromolar 4- Fluorescence values are converted to the absolute activity of α-Gal A by methyl umbellate form ketone standard curve, and are denoted as every milligram per hour The nanomole number (nmol/mg protein/hour) of the 4-MU dissociation of albumen.With untreated enzymatic activity standardized fluorescent number Value, uses to obtain the relative activity of α-Gal A, it is indicated with multiple.

Tentatively with the process in enzyme screening compounds library

Diluted compounds library (20 microlitres), makes 20 μM of its ultimate density；With the bottom for being dissolved in pH4.6 buffer (50 microlitres) Object 4- methylcoumarin base α-D- gala piperazine mutter glucosides (4-Methylumbelliferyl α-D-galactopyranoside, 20 Microlitre) and recombinant human α-Gal A (10 microlitres) is after evenly mixing, reacts 15 minutes in 37 DEG C.It is added and stops (every liter of solution 0.4 mole of K₂CO₃, pH 10.8), with 355 nanometers of excitation spectrum and 460 nanometers of diverging spectral measurement fluorescent value.Control Group is by HBTU, DIEA, acid compound library (total volume is 20 microlitres), substrate (20 microlitres), buffer (50 microlitres) and enzyme The mixture of (10 microlitres) compositions does not include acyl ammonia (amide) product.Blank control group (Blank) is then comprising being dissolved in pH The substrate (20 microlitres) and enzyme (10 microlitres) of 4.6 buffers (70 microlitres).Assessment inhibits effect, and is expressed as relative to control group Relative activity.

After removing compound by N215S sufferer lymphocyte, the process of follow-up analysis intracellular reactive enhancing

The Human Lymphocytes being mutated with N215S are planted with 20,000 cell quantity in 96 hole disks (coster), it cultivates 12-16 hours.After the inhibitor of certain concentration (0-50 μM) is added, persistently cultivate cell 4 days.Update 96 The cell culture fluid of hole disk is still further cultivated 0,2 or 4 day.After PBS washing cell 3 times, 50 microlitres of cell disintegration is delayed Fliud flushing (0.1M sodium phosphate/citrate buffer solution with 0.1%TX 100) is added in lymphocyte, after evenly mixing, with 4, The revolving speed of 000rpm is centrifuged 1 hour.The substrate solution of 20 microlitres of 20 microlitres of cellular breakdown products addition is taken, it includes be dissolved in 0.1M sodium phosphate/citrate buffer solution 8mM 4MU- α-D- gala piperazine is muttered glucosides and 150mM N- acetyl gala ammonia sugar, later It is reacted 1 hour in 37 DEG C.40 microlitres of stopping solution (sodium bicarbonate, pH 10.6) is added, then with 355 nanometers of excitation spectrum And 460 nanometers of diverging spectrum observes its fluorescent value.To be dissolved in every liter of 0.1M sodium phosphate/citrate buffer solution (20 microlitres) 0 nanomole is to 200 moles of methyl umbellate form ketone and 20 microlitres of 0.1M sodium phosphate/lemon acid buffering with 0.1%TX 100 Liquid establishes standard curve, uses the absolute activity that fluorescence values are converted to α-Gal A, is denoted as every milligram of egg per hour White nanomole.20 microlitres of cell point is determined with MicroBCA Protein assay set group (Pierce, Rockford, IL, USA) Solve the protein concentration in object.

The preparation of embodiment 1 and confirmation compound 9 to 37

1.1 synthesis compounds 9 to 24

It in the present embodiment, is according to method described in Tsou et al (Tetrahedron 2009,65,93), by 8 kinds Corresponding three-O- benzyl rings ring subunit amine-n-oxides (tri-O-benzyl cyclic nitrones) of Pyrrolizidine class chirality To prepare 16 kinds of non-natural stereoisomers (i.e. compound 9 to 24) of amido-DMDP (or ADMDP).Foundation uses ring subunit The chemical structure of amine-n-oxides, each ring subunit amine-n-oxides can be used as a kind of precursor, specifically have for two kinds with preparation 2,3- forward and 2, the class ADMDP product of 3- reverse geometry.Flow chart 1 provides the process of synthesis compound 17 and 18.Specifically For, the nucleophilicity TMSCN with height non-mirror image selectivity is added to the ring subunit amine-n-oxides for being dissolved in methanol in 50 DEG C After 25, primary product is its isomer 26 (78%, dr >=20/1), via reduction-N-protected (reductive-N- Protection) and O-Bn deprotection effect (O-Bn deprotection) can be further by compound 26 with high yield (60%) mode is converted to compound 17.On the other hand, (elimination- is handled in proper order using cancellation-reduction Reduction sequence) the C-2 nitrile group in compound 26 is converted and generates compound 27 (two steps into 38%).It connects , comprehensive hydrogenolysis compound 27 is the C2 epimer (yield 71%) of compound 17 with prepare compound 18.With with stream The similar method of journey Fig. 1 is prepared other isomers by corresponding ring subunit amine-n-oxides, and yield is about 30- 35%.

The process of 1. prepare compound 17 and 18 of flow chart

Condition and reagent: (a) TMSCN, MeOH, 50 DEG C, 10 hours, 78%；(b) 1) MsCl, Et₃N, THF, 20 minute, 2)NaBH₄, MeOH/THF, 13 hours, 38%；(c)Pd(OH)₂, conc.HCl, H₂, MeOH, 71%；(d) 1) Raney Ni, Boc₂O, H₂, MeOH, rt, 5 hours, 2) and conc.HCl, MeOH, reflux, 1 hour；3)Pd(OH)₂, conc.HCl, H₂, MeOH, rt, 10 hour, the yield of three steps are 60%.

Prepare compound 25. is by 2,3,5- tri--O- benzyl-L- core furanoses (2.4 grams, 5.8 mMs) and is dissolved in methanol The hydrochloric acid hydroxylamine (hydroxylamine hydrochloride, 3.2 grams, 46 mMs) and methoxylation sodium of (15 milliliters) (sodium methoxide, 4.3 milliliters, 23 mMs, in the concentration of methanol be 5.4M) hybrid reaction.Mixing is stirred at room temperature After object 5 hours, solvent is evaporated.Using EtOAc extraction leftover, and with H₂O washing, later with anhydrous MgSO₄It is dried, then Enriched product.CH will be dissolved in₂Cl₂Oxime (oxime) residue, the tert-butyl diphenyl chlorosilane (tert- of (7.5 milliliters) Butylchlorodiphenylsilane (TBDPSCl), 2.07 grams, 7.5 mMs) and imidazoles (imidazole, 0.6 gram, 9.0 mMs) be stirred at room temperature 2 hours after, with water (10 milliliters) extinguishing reaction.After salt water washing organic layer, utilization is anhydrous MgSO₄Desciccate is simultaneously concentrated.To be dissolved in CH₂Cl₂Mesyl chloride (the methanesulfonyl of (15 milliliters) Chloride, 0.67 milliliter, 8.7 mMs) and triethylamine (triethylamine, 1.2 milliliters, 8.7 mMs) processing silane (silane) mixture of residue reacts 3 hours in 0 DEG C, later with water (15 milliliters) extinguishing reaction mixture.It is washed with salt After washing organic layer, anhydrous MgSO is utilized₄Desciccate is simultaneously concentrated.At room temperature, stirring is thick in dry THF (15 milliliters) Intermediate product and TBAF (7.0 milliliters, concentration is 1M in THF) processed 30 minutes.Vacuum removes solvent, is dissolved in 80 DEG C of stirrings MeOH/H₂Residue, hydrochloric acid hydroxylamine (3.13 grams, 45 mMs) and the sodium bicarbonate (3.78 of O (4/1, volume ratio, 58 milliliters) Gram, 45 mMs) 24 hours.Vacuum removes solvent, then with CC (50%EtOAc is in hexane, silica gel) purification residues to prepare White solid-ring subunit amine-n-oxides 25 (1.09 grams, 2.61 mMs, 45%)；¹HNMR (600MHz, CDCl₃)δ4.09 (dd, 1H, J=3.3,9.2Hz), 4.15-4.19 (m, 2H), 4.37 (t, 1H, J=5.9Hz), 4.53-4.60 (m, 4H), 4.68 (s, 2H), 4.70 (d, 1H, J=11.8Hz), 6.91 (s, 1H), 7.24-7.35 (m, 15H)；¹³CNMR (150MHz, CDCl₃)δ 137.7,137.3,137.2,132.7,128.5,128.4,128.3,128.0,127.9,127.8,127.7,127.6, 127.5,74.5,74.0,73.4,73.0,72.4,66.5.HRMS calcd for [C₂₆H₂₇NO₄+Na]⁺440.1832, found 440.1843.

That prepare compound 26. will be dissolved in dry methanol (5 milliliters) includes compound 25 (420 milligrams, 1 mM) and three The mixture of methyl nitrile silicone (trimethylsilyl cyanide, 320 microlitres, 2.5 mMs) stirs 10 hours in 50 DEG C. Vacuum removes solvent, then with CC (20%EtOAc is in hexane, silica gel) purification residues to prepare pure white solid compound 26 (344 milligrams, 0.78 mM, 78%)；¹H NMR (600MHz, CDCl₃) δ 3.57 (dd, 1H, J=6.8,12.9Hz), 3.69 (dd, 1H, J=6.8,9.7Hz), 3.76 (dd, 1H, J=6.8,9.7Hz), 4.25 (t, 1H, J=5.5,5.4Hz), 4.32 (d, 1H, J=5.7Hz), 4.33 (t, 1H, J=5.5,5.3Hz), 4.47-4.53 (m, 3H), 4.63 (d, 1H, J=11.9Hz), 4.68-4.73 (m, 2H), 7.22-7.35 (m, 15H)；¹³C NMR (150MHz, CDCl₃) δ 141.6,141.5,140.7, 132.5,132.3,132.2,132.1,131.9,131.8,131.7,131.6,120.4,84.1,81.2,81.0,80.9, 79.8,72.9,71.3,64.3.HRMS calcd for [C₂₇H₂₈N₂O₄+Na]⁺467.1941 found 467.1959.

Prepare compound 17. in hydrogen environment, in anhydrous methanol (3 milliliters) mixed compound 26 (0.29 gram, 0.65 MM), Lei Shi nickel (Raney nickel, 0.05 gram) and di-tert-butyl dicarbonate (di-tert-butyl Dicarbonate, 700 milligrams, 3.25 mMs) and stir until it is completely dissolved.After 5 hours, vacuum removal solvent, then with CC (10%EtOAc is in hexane, silica gel) purification residues have boc-protected intermediate product with preparation.It is dissolved in 50 DEG C of reflux Methanol (3 milliliters) has boc-protected intermediate product and 37%HCl (1 milliliter).After 1 hour, palladium dydroxide (50 millis are added Gram), reaction mixture is again stirring in room temperature, hydrogen environment 10 hours.Reaction mixture is filtered using silicoglaserite, then with CC (25% aqueous NH₄OH (37%) is in 1- propyl alcohol, silica gel) it is purified, to prepare flaxen -17 (63.2 millis of oil product Gram, 0.39 mM, 60%).¹H NMR (600MHz, D₂O) δ 2.81 (dd, 1H, J=8.0,13.0Hz), 2.97 (dd, 1H, J =4.4,13.0Hz), 3.12-3.16 (m, 1H), 3.24 (d, 1H, J=3.6Hz), 3.60 (dd, 1H, J=6.6,11.2Hz), 3.74 (dd, 1H, J=6.6,11.2Hz), 3.90 (dd, 1H, J=4.4,8.0Hz), 4.11 (s, 1H)；¹³CNMR (150MHz, D₂O) 75.4 δ, 71.9,60.2,59.8,59.7,43.1.HRMS calcd for [C₆H₁₄N₂O₃+H]⁺163.1077, found 163.1083.

Prepare compound 27. is to be dissolved in the mesyl chloride (280 microlitres, 3.4 mMs) and triethylamine of THF (10 milliliters) (680 microlitres, 4.54 mMs) and compound 26 (1.09g, 2.27 mMs) react 20 minutes.After putting out reaction with water quenching, benefit Purified with EtOAc.With anhydrous MgSO₄After dry organic layer, enriched product.Stirring is dissolved in MeOH/THF in ice-water bath The mixture 13 comprising above-mentioned residue and sodium borohydride (210 milligrams, 5.2 mMs) of (1/1, volume ratio, 8 milliliters) is small When.After removing solvent, reaction mixture is extracted with EtOAc, and with salt water washing.With anhydrous MgSO₄Dry organic layer is simultaneously concentrated Afterwards, purified using CC (25%EtOAc is in hexane, silica gel), with prepare white solid-pure 27 (391 milligrams, 0.91 mmoles You, 38%).¹H NMR (600MHz, CDCl₃) δ 3.41 (dd, 1H, J=6.2,10.3Hz), 3.59 (dd, 1H, J=7.3, 9.2Hz), 3.72 (dd, 1H, J=6.2,9.2Hz), 4.02 (m, 2H), 4.19 (d, 1H, J=7.3Hz), 4.45 (d, 1H, J= 11.7Hz), 4.50 (d, 1H, J=11.7Hz), 4.60-4.65 (m, 2H), 4.70 (d, 1H, J=11.9Hz), 4.95 (d, 1H, J =11.9Hz), 7.24-7.40 (m, 15H)；¹³C NMR (150MHz, CDCl₃) δ 138.3,137.9,137.0,128.3, 128.1,128.0,127.8,127.6,127.5,127.5,127.4,127.2,119.2,80.2,76.6,73.2,73.1, 72.6,70.1,58.5,48.5.HRMScalcd for [C₂₇H₂₈N₂O₃+H]⁺429.2173 found 429.2181.

Prepare compound 18. will contain compound 27 (69 milligrams, 0.16 mM) and palladium dydroxide in hydrogen environment (200mg) object and methanol (1 milliliter) are mixed and stirred for 24 hours, make to be completely dissolved.After being filtered with silicoglaserite, it is concentrated and with CC (11.1% aqueous NH₄OH (37%) is in 1- propyl alcohol, silica gel) purifying filtration product, to prepare (18.5 millis of faint yellow solid -18 Gram, 0.11 mM, 71%).¹H NMR (600MHz, D₂O) δ 3.45 (dd, 1H, J=5.8,13.8Hz), 3.59 (dd, 1H, J =6.7,13.8Hz), 3.77-3.80 (m, 1H), 3.91 (dd, 1H, J=8.4,12.2Hz), 3.99 (dd, 1H, J=4.9, 12.2Hz), 4.06 (q, 1H, J=6.5Hz), 4.46 (t, 1H, J=4.6Hz), 4.62 (dd, 1H, J=4.3,7.0Hz)；¹³C NMR (150MHz, D₂O) 70.3 δ, 69.8,61.9,57.7,56.0,37.2.HRMS calcd for [C₆H₁₄N₂O₃+Na]⁺ 185.0897.found 185.0901.

The flow chart of preparation 19

Condition and reagent: the method for [1] Tsou et al., Tetrahedron 2009,65,93；(a) TMSCN, MeOH, 50 DEG C, 7 hours, 83%；(b) 1) Raney Ni, Boc₂O, H₂, MeOH, rt, 12 hours；2) conc.HCl, MeOH, reflux, 1 Hour；3)Pd(OH)₂, conc.HCl, H₂, MeOH, the yield of three steps is 51%.

Prepare compound S1. is according to process prepare compound S1 similar with prepare compound 25, the difference is that will Mesylation reaction (mesylation) is replaced into iodination reaction (iodination).Flow back be dissolved in toluene (15 milliliters) include Silane residue (3.2 grams, 4 mMs), triphenyl phasphine (triphenylphosphine, 3.15 grams, 12.0 mMs), imidazoles Mixture 1 hour of (0.82 gram, 12.0 mMs) and iodine (2.03 grams, 8.0 mMs), after filtering reaction mixture, with EtOAc washing.After benefit is washed with brine organic layer, with anhydrous MgSO₄Product is dried and concentrated.Crude intermediate product is dissolved in first Benzene (20 milliliters), and TBAF (4.8 milliliters, 1M is in THF) are added.In 110 DEG C after back flow reaction 30 minutes, mixture is concentrated, and It is purified with CC (25%EA is in hexane, silica gel), prepares syrupy shape product-ring subunit amine-n-oxide S 1 (0.53 accordingly Gram, 1.27 mMs, 32%).¹H NMR (600MHz, CDCl₃) δ 3.62 (dd, 1H, J=2.4,10.6Hz), 4.10-4.11 (m, 1H), 4.14 (dd, 1H, J=2.5,10.7Hz), 4.41 (d, 1H, J=11.9Hz), 4.46 (dd, 1H, J=4.7, 6.0Hz), 4.55 (dd, 2H, J=3.5,11.7Hz), 4.59-4.70 (m, 4H), 6.93 (s, 1H), 7.22-7.37 (m, 15H) ；¹³C NMR (150MHz, CDCl₃) δ 137.6,137.4,137.2,133.4,128.63 (x2), 128.58 (x2), 128.5 (x2), 128.21,128.16 (x4), 128.1 (x2), 127.9,127.7 (x2), 76.4,75.4,75.1,73.5,72.5, 72.1,64.8.HRMS calcd for [C₂₆H₂₇NO₄+H]⁺418.2013 found 418.2020.

Prepare compound S2. is solid come the white of prepare compound S2 (83%) according to the reaction of prepare compound 26 by S1 Body.¹H NMR (600MHz, CDCl₃) δ 3.31 (q, 1H, J=4.6Hz), 3.49-3.54 (m, 2H), 3.83 (t, 1H, J= 5.2Hz), 4.06-4.11 (m, 2H), 4.44-4.53 (m, 3H), 4.58 (d, 1H, J=11.8Hz), 4.63 (d, 1H, J= 11.8Hz), 4.68 (d, 1H, J=11.7Hz), 7.24-7.35 (m, 15H)；¹³CNMR (150MHz, CDCl₃) δ 137.5, 137.4,136.7,128.5 (x2), 128.41 (x2), 128.38 (x2), 128.2,128.1 (x2), 128.0 (x2), 127.9, 127.83,127.81 (x2), 118.5,77.3,75.1,73.3,72.7,72.1,71.9,67.9,61.8.HRMS calcd for[C₂₇H₂₈N₂O₃+H]⁺445.2122 found 445.2130.

Prepare compound 19. prepares the reaction of chemical combination 17 as starting material foundation by S2 come prepare compound 19 (51%) Faint yellow solid..¹H NMR (600MHz, D₂O) δ 2.75 (dd, 1H, J=7.6,13.1Hz), 2.93 (dd, 1H, J=5.1, 13.1Hz), 3.07-3.11 (m, 2H), 3.59 (dd, 1H, J=5.8,11.7Hz), 3.67 (dd, 1H, J=4.7,11.7Hz), 3.76 (t, 1H, J=6.2), 3.86 (t, 1H, J=5.7Hz)；¹³CNMR (150MHz, D₂O) 73.5 δ, 72.0,63.6,62.4, 62.3,43.0.HRMS calcd for [C₆H₁₄N₂O₃+H]⁺163.1077 found 163.1071.

Prepare compound 9. is prepared as starting material according to above-mentioned steps faint yellow by chiral ring subunit amine-n-oxides Solid chemical compound -9 (45%)¹H NMR (600MHz, D₂O) δ 3.18 (dd, 2H, J=8.4,13.3Hz), 3.31 (dd, 1H, J= 4.8,13.3Hz), 3.40-3.43 (m, 1H), 3.73 (dd, 1H, J=6.1,11.9Hz), 3.81 (dd, 1H, J=3.9, 12.1Hz), 3.92-3.95 (m, 2H)；¹³C NMR (150MHz, D₂O) 78.6,76.4,62.1,60.5,58.0,41.3.HRMS δ calcd for[C₆H₁₄N₂O₃+H]⁺163.1077, found163.1071.

Prepare compound 10. prepares chemical combination according to above-mentioned steps as starting material by chiral ring subunit amine-n-oxides The faint yellow solid of object 10 (25%)¹H NMR (600MHz, D₂O) δ 3.09 (dd, 1H, J=6.3,13.1Hz), 3.13 (q, 1H, J=5.5Hz), 3.24 (dd, 1H, J=6.0,13.1Hz), 3.59 (q, 1H, J=6.1Hz), 3.64 (dd, 1H, J=6.5, 11.6Hz), 3.74 (dd, 1H, J=4.5,11.6Hz), 3.90 (dd, 1H, J=3.8,5.3Hz), 4.20 (dd, 1H, J=3.8, 5.8Hz)；¹³C NMR (150MHz, D₂O) 77.9 δ, 76.8,64.0,62.2,55.9,39.2.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077 found 163.1074.

Prepare compound 11. prepares chemical combination according to above-mentioned steps as starting material by chiral ring subunit amine-n-oxides The faint yellow solid of object 11 (43%).¹H NMR (600MHz, D₂O) δ 3.24 (dd, 1H, J=6.9,13.8Hz), 3.35 (dd, 1H, J=6.6,13.8Hz), 3.68 (dd, 1H, J=8.8,12.1Hz), 3.79 (dd, 1H, J=4.7,12.1Hz), 3.80- 3.90 (m, 2H), 4.28 (d, 1H, J=3.6Hz), 4.32 (d, 1H, J=3.6Hz)；¹³C NMR (150MHz, D₂O)δ75.8 (x2), 61.8,58.8,57.3,38.0.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077 found 163.1078.

Prepare compound 12. prepares chemical combination according to above-mentioned steps as starting material by chiral ring subunit amine-n-oxides The faint yellow solid of object 12 (27%).¹H NMR (600MHz, D₂O) δ 3.24 (dd, 1H, J=7.6,13.3Hz), 3.31 (dd, 1H, J=5.3,13.3Hz), 3.43-3.45 (m, 1H), 3.61 (dd, 1H, J=5.2,12.2Hz), 3.73 (dd, 1H, J= 7.2,11.5Hz), 3.83 (dd, 1H, J=5.3,11.5Hz), 4.02 (t, 1H, J=3.2Hz), 4.20 (dd, 1H, J=3.0, 4.9Hz)；¹³C NMR (150MHz, D₂O) 78.8 δ, 75.6,61.5,60.8,59.3,41.0.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077 found 163.1069.

Prepare compound 13. prepares chemical combination according to above-mentioned steps as starting material by chiral ring subunit amine-n-oxides The faint yellow solid of object 13 (55%).¹H NMR (600MHz, D₂O) δ 3.54 (m, 2H), 3.81 (dd, 1H, J=4.0,9.0Hz), (3.86 m, 2H), 3.95 (dd, 1H, J=3.8,12.6Hz), 4.22-4.27 (m, 2H)；¹³C NMR (150MHz, D₂O) 72.5 δ, 70.2,65.9,58.8,58.4,38.65.HRMS calcd for [C₆H₁₄N₂O₃+H]⁺163.1077 found 163.1078.

Prepare compound 14. prepares chemical combination according to above-mentioned steps as starting material by chiral ring subunit amine-n-oxides The faint yellow solid of object 14 (31%).¹H NMR (600MHz, D₂O) δ 3.46 (dd, 1H, J=6.5,13.8Hz), 3.61 (dd, 1H, J=6.7,13.8Hz), 3.69-3.72 (m, 1H), 3.85 (dd, 1H, J=5.9,12.7Hz), 3.97-4.02 (m, 2H), 4.32 (dd, 1H, J=3.8,8.7Hz), 4.43 (t, 1H, J=3.5Hz)；¹³C NMR (150MHz, D₂O) 71.2 δ, 70.1, 62.4,57.9,57.7,36.1.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077 found 163.1073.

Prepare compound 15. prepares chemical combination according to above-mentioned steps as starting material by chiral ring subunit amine-n-oxides The faint yellow solid of object 15 (47%).¹H NMR (600MHz, D₂O) δ 3.11 (dd, 1H, J=8.5,13.1Hz), 3.24 (dd, 1H, J=4.8,13.1Hz), 3.35-3.41 (m, 2H), 3.68 (dd, 1H, J=7.0,11.5Hz), 3.82 (dd, 1H, J= 6.1 11.5Hz), 4.03 (dd, 1H, J=4.0,8.7Hz), 4.17 (t, 1H, J=3.8Hz)；¹³C NMR (150MHz, D₂O)δ 75.4,71.5,60.3,59.7,57.7,42.0.HRMS calcd for [C₆H₁₄N₂O₃+H]⁺163.1077, found 163.1073.

Prepare compound 16. prepares compound 16 (15%) according to above-mentioned steps by chiral ring subunit amine-n-oxides Faint yellow solid.¹H NMR (600MHz, D₂O) δ 3.16 (dd, 1H, J=5.3,11.6Hz), 3.24 (dd, 1H, J=5.9, 13.5Hz), 3.44 (d, 1H, J=5.2Hz), 3.64-3.69 (m, 2H), 3.82 (dd, 1H, J=5.3,11.5Hz), 4.3 (t, 1H, J=4.7Hz), 4.45 (dd, 1H, J=4.6,7.2Hz)；¹³C NMR (150MHz, D₂O) 71.4,70.7,60.0,55.0 δ (2), 39.0.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077 found 163.1074.

Prepare compound 20. prepares compound 20 (43%) according to above-mentioned steps by chiral ring subunit amine-n-oxides Faint yellow solid.¹H NMR (600MHz, D₂O) δ 3.46 (dd, 1H, J=6.5,13.8Hz), 3.61 (dd, 1H, J=6.7, 13.8Hz), 3.69-3.72 (m, 1H), 3.85 (dd, 1H, J=5.9,12.7Hz), 3.97-4.02 (m, 2H), 4.32 (dd, 1H, J=3.8,8.7Hz), 4.43 (t, 1H, J=3.5Hz)；¹³CNMR (150MHz, D₂O) 71.2 δ, 70.1,62.4,57.9,57.7, 36.1.HRMS calcd for[C₆H₁₄N₂O+H]⁺163.1077 found 163.1073.

Prepare compound 21HCl. prepares compound 21 according to above-mentioned steps by chiral ring subunit amine-n-oxides (36%) faint yellow solid.¹H NMR (600MHz, D₂O) δ 3.48 (dd, 1H, J=6.9,13.8Hz), 3.57 (dd, 1H, J= 6.6,13.8Hz), 3.90 (dd, 1H, J=8.8,12.1Hz), 3.98 (dd, 1H, J=4.7,12.1Hz), 4.03-4.06 (m, 1H), 4.14-4.17 (m, 1H), 4.39 (d, 1H, J=3.6Hz), 4.41 (d, 1H, J=3.6Hz)；¹³C NMR (150MHz, D₂O) 74.6 δ, 74.2,63.7,58.2,57.3,36.0.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077, found 163.1078.

Prepare compound 22. prepares compound 22 (27%) according to above-mentioned steps by chiral ring subunit amine-n-oxides Faint yellow solid.¹H NMR (600MHz, D₂O) δ 3.24 (dd, 1H, J=7.6,13.3Hz), 3.31 (dd, 1H, J=5.3, 13.3Hz), 3.43-3.45 (m, 1H), 3.61 (dd, 1H, J=5.2,12.2Hz), 3.73 (dd, 1H, J=7.2,11.5Hz), 3.83 (dd, 1H, J=5.3,11.5Hz), 4.02 (t, 1H, J=3.2Hz), 4.20 (dd, 1H, J=3.0,4.9Hz)；¹³C NMR (150MHz, D₂O) 78.8 δ, 75.6,61.5,60.8,59.3,41.0.HRMS calcd for [C₆H₁₄N₂O+H]⁺163.1077 found 163.1069.

Prepare compound 23. is by chiral ring subunit amine-n-oxides according to above-mentioned steps prepare compound 23 (46%) Faint yellow solid.¹H NMR (600MHz, D₂O) δ 3.03 (m, 2H), 3.18 (dd, 1H, J=4.3,12.9Hz), 3.24 (m, 1H), 3.64 (dd, 1H, J=5.7,11.4Hz), 3.71 (dd, 1H, J=3.4,11.5Hz), 3.78-3.83 (m, 2H)；¹³C NMR (150MHz, D₂O) 79.2 δ, 77.1,61.7,61.1,57.9,42.0.HRMS calcd for [C₆H₁₄N₂O₃+H]⁺ 163.1077 found 163.1071.

Prepare compound 24. is by chiral ring subunit amine-n-oxides according to above-mentioned steps prepare compound 24 (37%) Faint yellow solid.¹H NMR (600MHz, D₂O) δ 3.51 (dd, 1H, J=6.5,13.8Hz), 3.60 (dd, 1H, J=6.5, 13.8Hz), 3.67-3.70 (m, 1H), 3.85 (dd, 1H, J=8.6,12.1Hz), 3.97 (dd, 1H, J=4.8,12.1Hz), 4.08-4.11 (m, 1H), 4.13 (s, 1H), 4.36 (s, 1H)；¹³C NMR (150MHz, D₂O) 75.7 δ, 74.7,67.9,59.3, 58.6,35.6.HRMS calcd for [C6H14N2O+H]⁺163.1077 found 163.1072.

1.2 synthesis compounds 28 and 29

Method according to flow chart 2 distinguishes prepare compound 28 and 29.Specifically, using hydrogenolysis by flow chart 1 Ring subunit amine-n-oxides 25 carry out prepare compound 28, and reacted by selectively acylating and compound synthesized by compound 17 29。

Flow chart 2

Condition and reagent: (a) MeOH, Pd (OH)₂, H_2(g), 79% (b) Ac₂O, MeOH/H₂O, rt, 4 hour, 56%.

Prepare compound 28. in hydrogen environment, be stirred methanol (1.5 milliliters) and compound 25 (81 milligrams, 0.19 MM) and palladium dydroxide (30 milligrams) 24 hours, until being completely dissolved.After crossing Lu with silicoglaserite, it is concentrated and with CC (25% water Property NH₄OH (37%) is in 1- propyl alcohol, silica gel) purifying filtration product, (20.0 in the least for the faint yellow solid of prepare compound 28 accordingly Gram, 0.15 mM, 79%).¹H NMR (600MHz, D₂O) δ 3.23 (dd, 1H, J=7.3,12.1Hz), 3.55 (dd, 1H, J =7.3,12.1Hz), 3.74-3.77 (m, 2H), 3.91 (dd, 1H, J=8.6,12.2Hz), 4.01 (dd, 1H, J=4.8, 12.2Hz), 4.37 (t, 1H, J=4.2Hz), 3.86 (td, 1H, J=4.1,7.4Hz)；¹³C NMR (150MHz, D₂O) 70.0 δ, 69.8,62.4,57.6,47.0.HRMS calcd for [C₅H₁₁NO₃+H]⁺134.0812 found 134.0813.

Prepare compound 29. is stirred H at room temperature₂O/MeOH solution (1/3, volume ratio, 400 microlitres) and compound 17 (10.0 milligrams, 0.06 mM) and acetic anhydride (acetic anhydride, 15.0 microlitres, 0.15 mM) 4 hours, until It is completely dissolved.With Dowex 550A (OH^-) anion exchange resin neutralization reaction mixture.After filtering mixture, with MeOH (2 Milliliter) washing.Thickening filtration product, and with CC (12.5% aqueous NH₄OH (37%) is in propyl alcohol, silica gel) purification residues, with Prepare yellow syrup product -29 (6.9 milligrams, 0.03 mM, 56%).¹H NMR (600MHz, D₂O) δ 2.04 (s, 3H), 3.57 (dd, 1H, J=8.1,15.8Hz), 3.66 (m, 2H), 3.77 (m, 1H), 3.88 (dd, 1H, J=8.4,12.0Hz), 3.97 (dd, 1H, J=5.2,12.0Hz), 4.20 (dd, 1H, J=3.9,8.9Hz), 4.31 (t, 1H, J=3.5Hz)；¹³C NMR (150MHz, D₂O) 176.0 δ, 72.7,69.9,61.8,60.4,57.6,39.1,21.6.HRMS calcd for [C₈H₁₆N₂O₄+ H]⁺205.1183 found 205.1184.

1.3 synthesis compounds 30 to 37

In the present embodiment, 60 in precursor compound library 5 and compound library 6 are screened according to the method for flow chart 3 and 4 Kind compound；5 kinds of potentiality compounds are respectively synthesized to synthesize the method for compound 33 according to described in flow chart 5 later (i.e. compound 33 to 37).

Flow chart 3

Condition and reagent: (a) TMSCN, MeOH, 50 DEG C, 10 hours, 78%；(b) 1) MsCl, Et₃N, THF, 20 minute, 2)NaBH₄, MeOH/THF, 13 hours, 38%；3)Pd(OH)₂, conc.HCl, H₂, MeOH, 71%；(c) 1) Raney Ni, Boc₂O, H₂, MeOH, rt, 5 hours, 2) and conc.HCl, MeOH, reflux, 1 hour；3)Pd(OH)₂, conc.HCl, H₂, MeOH, Rt, 10 hours, the yield of three steps was 60%.

Flow chart 4

Compound 17 and 18 has common (3R, 4S, 5R) configuration (flow chart 3), prepares two using those compounds Compound library, i.e. compound library 5 and compound library 6 (flow chart 4), wherein be to except compound 17 and 18-aminomethyl group into Row, which has, replaces multifarious parallel projects, is to be dissolved in the HBTU of DMF (1.5 equivalent), HOBt (1.5 equivalent) and DIEA (3 Equivalent) after activating carboxy acid, and amido bond knot is formed by one of substituted carboxylic acid that 60 kinds of acids are selected at random.24 hours Afterwards, analysis reactant can find that amine 17 and 18 has been totally consumed, and synthesize the specific product of significant content.It is not required to carry out other Purification step is directly tested with the relevant inhibition of α-Gal A enzyme to assess the 5 (source of precursor compound library with 60 kinds of compounds From compound 17) and compound library 6 (being originated from compound 18), assessing concentration is 20 μM.

Results of preliminary screening generates 5 kinds of potentiality compounds, can produce effective inhibitory activity (> 60% inhibits reaction) (result is not shown).In comparison, it is not found to have the inhibitor of potentiality then in compound library 6 (result is not shown).Those hairs It now points out, the configuration of the position C2 will affect inhibition effect.In order to further confirm those inhibitor, recombines this 5 kinds and dive Power compound, i.e. compound 33-37.Flow chart 5 provides the illustrative process to synthesize compound 33.

Flow chart 5

Reagent and reaction: (a) SmI₂, HOAc, THF, 0 DEG C of to rt, 1 hour, 70%；(b)Boc₂O, DIEA, CH₂Cl₂, 12 hours；(c) Raney Ni, H₂, MeOH, 3 hours, two steps were 50% (d) 1) RCOOH, EDC, DMAP, CH₂CL₂, 3 hours, 2)Pd(OH)₂, H₂, HCl, 8 hours, two steps were 50%.

Prepare compound 25. makes 2,3,5- tri--O- benzyl-L- core furanoses (2.4 grams, 5.8 mMs) and is dissolved in methanol The hydrochloric acid hydroxylamine (hydroxylamine hydrochloride) (3.2 grams, 46 mMs) and methoxylation sodium (4.3 of (15 milliliters) Milliliter, is reacted in the concentration of methanol for 5.4M) is common by 23 mMs.After mixture being stirred at room temperature 5 hours, solvent is evaporated.Benefit With EtOAc extraction leftover, and with H₂O washed product, later with anhydrous MgSO₄It is dried, is concentrated.It is stirred at room temperature It is dissolved in CH₂Cl₂Oxime residue, tert-butyl diphenyl chlorosilane (TBDPSCl, 2.07 grams, 7.5 mMs) and the miaow of (7.5 milliliters) Azoles (0.6 gram, 9.0 mMs) is after 2 hours, with water (10 milliliters) extinguishing reaction.After benefit is washed with brine organic layer, with anhydrous MgSO₄It is dry, and be concentrated.To be dissolved in CH₂Cl₂Mesyl chloride (0.67 milliliter, the 8.7 mMs) Ji Sanyi of (15 milliliters) Amine (1.2 milliliters, 8.7 mMs) in 0 DEG C processing silane residual mixture 3 hours, then with water (15 milliliters) extinguishing react mix Object.With salt water washing organic layer, anhydrous MgSO is utilized₄After drying, it is concentrated.It is stirred at room temperature and is dissolved in dry THF (15 millis Rise) crude intermediate product and TBAF (7.0 milliliters, 1M is in THF) 30 minutes.After vacuum removes solvent, it is dissolved in 80 DEG C of stirrings MeOH/H₂Residue, hydrochloric acid hydroxylamine (3.13 grams, 45 mMs) and the sodium bicarbonate of O (4/1, volume ratio, 58 milliliters) (3.78g, 45 mMs) 24 hours.After vacuum removes solvent, with CC (50%EtOAc is in hexane, silica gel) purification residues, with Prepare white solid-ring subunit amine-n-oxides 25 (1.09 grams, 2.61 mMs, 45%).¹H NMR (600MHz, CDCl₃)δ 4.09 (dd, 1H, J=3.3,9.2Hz), 4.15-4.19 (m, 2H), 4.37 (t, 1H, J=5.9Hz), 4.53-4.60 (m, 4H), 4.68 (s, 2H), 4.70 (d, 1H, J=11.8Hz), 6.91 (s, 1H), 7.24-7.35 (m, 15H)¹³C NMR (150MHz, CDCl₃) δ 137.7,137.3,137.2,132.7,128.5,128.4,128.3,128.0,127.9,127.8,127.7, 127.6,127.5,74.5,74.0,73.4,73.0,72.4,66.5.HRMS calcd for [C₂₆H₂₇NO₄+Na]⁺ 440.1832, found440.1843.

Prepare compound 26. is stirred anhydrous methanol (5 milliliters), compound 25 (420 milligrams, 1 mM) in 50 DEG C And mixture 10 hours of trimethyl nitrile silicone (320 microlitres, 2.5 mMs).After vacuum removes solvent, with CC (20%EtOAc In hexane, silica gel) purification residues, to prepare white solid-pure 26 (344 milligrams, 0.78 mM, 78%)；¹H NMR (600MHz, CDCl₃) δ 3.57 (dd, 1H, J=6.8,12.9Hz), 3.69 (dd, 1H, J=6.8,9.7Hz), 3.76 (dd, 1H, J=6.8,9.7Hz), 4.25 (t, 1H, J=5.5,5.4Hz), 4.32 (d, 1H, J=5.7Hz), 4.33 (t, 1H, J=5.5, 5.3Hz), 4.47-4.53 (m, 3H), 4.63 (d, 1H, J=11.9Hz), 4.68-4.73 (m, 2H), 7.22-7.35 (m, 15H) ；¹³CNMR (150MHz, CDCl₃) δ 141.6,141.5,140.7,132.5,132.3,132.2,132.1,131.9,131.8, 131.7,131.6,120.4,84.1,81.2,81.0,80.9,79.8,72.9,71.3,64.3.HRMS calcd for [C₂₇H₂₈N₂O₄+Na]⁺467.1941 found 467.1959.

The compound 26 (800 milligrams, 1.8 mMs) and 75 for being dissolved in THF (0.1M) is stirred at room temperature in prepare compound 30. Milliliter means of samarium iodide (I) and HOAc (2.2 milliliters) totally 1 hour.After the filtering of silico-calcium slabstone, solvent is removed, then with EtOAc And Na₂S₂O_{3 (liquid)}Extract reaction mixture.With MgSO₄After drying organic layer and being concentrated, using CC, (25%EtOAc is in oneself Alkane, silica gel) it purifies to obtain colorless oil as product-pure products (540 milligrams, 0.91 mM, 70%).¹H NMR (600MHz, CDCl₃) δ 3.55-3.58 (m, 2H), 3.65 (dd, 1H, J=9.2,12.2Hz), 4.07-4.09 (m, 2H), 4.26 (dd, 1H, J =4.1,6.6Hz), 4.51-4.53 (m, 2H), 4.58 (d, 1H, J=11.6Hz), 4.65-4.68 (m, 2H), 4.77 (s, 1H, 11.6Hz), 7.32-7.40 (m, 15H)¹³CNMR (150MHz, CDCl₃) δ 141.8,141.7,140.8,132.5,132.3, 132.2,132.0,131.8,131.7,131.7,131.7,131.6,124.4,87.5,81.2,81.0,80.9,80.8, 72.7,62.9,53.9.HRMS calcd for [C₂₇H₂₈N₂O₃+H]⁺429.2173 found 429.2174.

Compound 30 (540 milligrams, 1.26 mMs), the Boc for being dissolved in DCM is stirred at room temperature in prepare compound 32.₂O (550 milligrams) and triethylamine (0.17 milliliter) 24 hours.After concentrated reaction solution, to be dissolved in the Lei Shi Ni and H of methanol₂(g) also Former cyanide group 3 hours.Concentration mixture, and purified with CC (6%MeOH is in DCM, silica gel), to obtain colorless oil Product-pure products (340 milligrams, 0.63 mM, 50%).

¹H NMR (600MHz, CDCl₃) δ 1.43 (d, 9H), 2.53 (dd, 1H, 8.5, J=17.9Hz), 2.87-2.97 (m, 1H), 3.59 (d, 1H, J=8.9Hz), 3.74-3.80 (m, 1H), 3.93 (d, 1H, J=3.8Hz), 4.14-4.20 (m, 3H), 4.45-4.80 (m, 6H), 7.24-7.33 (m, 15H)¹³C NMR (150MHz, CDCl₃) δ 154.3,138.5,138.3, 138.2,128.3,128.2,128.0,127.8,127.6,127.5,127.3,127.2,127.1,79.9,79.5,77.7, 73.1,72.3,71.9,70.2,64.3,58.3,42.6,28.4.HRMS calcd for [C₃₂ H₄₁ N₂ O₅+H]⁺ 533.3010, found533.3015.

Prepare compound 33., which is stirred at room temperature, is dissolved in CH₂Cl₂Compound 32 (33.3 milligrams, 0.0625 mM), EDCHCl (35.9 milligrams, 0.1875 mM), 4- dimethylamino pyridine (4-dimethylamnopyridine, 7.64 millis Gram, 0.0625 mM) and hexamethylene valeric acid (cyclohexanepentanoic acid, 17.27 milligrams, 0.0937 mM) 3 Hour.After putting out reaction with water quenching, CH is utilized₂Cl₂Extract product.By anhydrous MgSO₄Dry organic layer.With CC (33%EtOAc In hexane, silica gel) purification residues, to obtain amide intermediate product.In 50 DEG C flow back be dissolved in methanol amide intermediate product and 37%HCl.After 1 hour, palladium dydroxide (50 milligrams) is added, it is small to be persistently stirred to react mixture 10 in room temperature, hydrogen environment When.After filtering reaction mixture with silicoglaserite and be concentrated, CC (4.5%H is utilized₂O and 28%MeOH are in CHCl₃, silica gel) and it purified Product is filtered, to obtain faint yellow oil product -33 (9.87 milligrams, 0.03 mM, 48% yield).¹H NMR (600MHz, D₂O) δ 1.04-1.24 (m, 11H), 1.49-1.63 (m, 6H), 2.18 (s, 2H), 3.09 (s, 1H), 3.22-3.24 (m, 2H), (3.35 d, 1H, J=11.6Hz), 3.52-3.55 (m, 1H), 3.70-3.67 (m, 1H), 3.82 (s, 1H), 4.08 (s, 1H)；¹³C NMR (150MHz, D₂O) 176.4 δ, 75.2,71.9,60.6,59.9,59.7,41.7,37.4,37.1,36.0,33.2,26.6, 26.4,26.3,25.9.HRMS calcd for [C₁₇H₃₂N₂O₄+H]⁺329.2435 found 329.2435.

Prepare compound 34., which is stirred at room temperature, is dissolved in CH₂Cl₂Compound 32 (53.9 milligrams, 0.1 mM), EDC HCl (57.5 milligrams, 0.3 mM), 4- dimethylamino pyridine (12.2 milligrams, 0.1 mM) and 3- (3- anisyl)-the third Acid (3- (3-methoxyphenyl)-propionic acid, 27 milligrams, 0.15 mM) 3 hours.With H₂O extinguishing reaction Afterwards, CH is utilized₂Cl₂It is extracted.By anhydrous MgSO₄Dry organic layer is simultaneously concentrated.With CC (33%EtOAc in hexane, Silica gel) purification residues, to prepare amide intermediate product.It flows back in 50 DEG C and is dissolved in the amide intermediate product and 37%HCl of methanol. After 1 hour, palladium dydroxide (50 milligrams) are added and are persistently stirred to react mixture 10 hours in room temperature, hydrogen environment.With silicon After calcium stone filters reaction mixture and is concentrated, CC (4.5%H is utilized₂O and 28%MeOH are in CHCl₃, silica gel) and it is purified, with system Standby colorless oil as product -34 (14.52 milligrams, 0.047 mM, 47.1% yield).¹H NMR (600MHz, D₂O)δ263- 2.70 (m, 2H), 2.96 (t, 2H), 3.03 (m, 1H), 3.19 (s, 1H), 3.32 (dd, 1H, J=6.0,14.6Hz), 3.45 (dd, 1H, J=3.3,14.6Hz), 3.58 (dd, 1H, J=4.0,8.7Hz), 3.64 (dd, 1H, J=7.1,11.2Hz), 3.77 (dd, J=6.0,11.2Hz), 3.86 (s, 3H), 4.03 (t, 1H, J=3.3Hz), 6.91-6.95 (m, 3H), 7.35 (t, 1H, J =7.5Hz)；¹³C NMR (150MHz, D₂O) 176.5 δ, 158.9,142.2,129.9,121.4,121.2,114.2,111.9, 73.5,71.3,59.9,59.7,55.2,40.0,36.8,31.1.HRMS calcd for [C₁₆H₂₄N₂O₅+H]⁺325.1763 found 325.1764.

Prepare compound 35., which is stirred at room temperature, is dissolved in CH₂Cl₂Compound 32 (40.1 milligrams, 0.075 mM), EDCHCl (43 milligrams, 0.225 mM), 4- dimethylamino pyridine (9.16 milligrams, 0.075 mM) and 3- (2,4,5- Trimethoxy-phenyl) and-propionic acid (3- (2,4,5-trimethoxy-phenyl)-propionic acid, 27 milligrams, 0.1125 milli Mole) 3 hours.With H₂After O extinguishing reaction, CH is utilized₂Cl₂It is extracted.By anhydrous MgSO₄Dry organic layer simultaneously gives dense Contracting.With CC (33%EtOAc is in hexane, silica gel) purification residues, to obtain amide intermediate product.Methanol is dissolved in 50 DEG C of reflux Amide intermediate product and 37%HCl.After 1 hour, it is added palladium dydroxide (50 milligrams), in room temperature, hydrogen environment, persistently stirs Mix reaction mixture 10 hours.After filtering reaction mixture with silicoglaserite and be concentrated, CC (4.5%H is utilized₂O and 28%MeOH in CHCl₃, silica gel) and it is purified, to obtain faint yellow solid -35 (15.08 milligrams, 0.039 mM, 52% yield).¹H NMR (600MHz, D₂O) δ 2.49-2.54 (m, 1H), 2.55-2.60 (m, 1H), 2.77-2.83 (m, 2H), 3.06 (t, 1H, J= 7.6Hz), 3.22-3.37 (m, 2H), 3.47 (d, 1H, J=12.9Hz), 3.63 (dd, 1H, J=3.5,8.3Hz), 3.68 (dd, 1H, J=8.3Hz, 11.6Hz), 3.74 (s, 3H), 3.75 (d, 1H, J=5.7Hz), 3.77 (s, 3H), 3.79 (s, 3H), 3.97 (t, 1H, J=3.5Hz), 6.70 (s, 1H), 6.81 (s, 1H)；¹³C NMR (150MHz, D₂O) 177.6 δ, 151.7,147.5, 141.9 120.2,114.5,98.8,72.5,70.4,61.2,60.2,58.3,56.7,56.4,55.9,39.0,35.5, 25.5.HRMS calcd for[C₁₈ H₂₈ N₂ O₇+H]⁺385.1975 found 385.1978.

Prepare compound 37., which is stirred at room temperature, is dissolved in CH₂Cl₂Compound 32 (35 milligrams, 0.0657 mM), EDC HCl (37.8 milligrams, 0.197 mM), 4- dimethylamino pyridine (8.03 milligrams, 0.0657 mM) and trans- -4- (three Fluoro-2-methyl) cinnamic acid (trans-4- (trifluoro-methyl) cinnamic acid, 21.3 milligrams, 0.131 mM) 3 Hour.With H₂After O extinguishing reaction, CH is utilized₂Cl₂It is extracted.By anhydrous MgSO₄Dry organic layer is simultaneously concentrated.With CC (33%EtOAc is in hexane, silica gel) purification residues, to obtain amide intermediate product.CH is dissolved in -78 DEG C of reflux₂Cl₂Acyl Amine intermediate product and BBr₃(32 microlitres) 2 hours.With EtOH extinguishing reaction mixture and after being concentrated, CC (4.5%H is utilized₂O and 28%MeOH is in CHCl₃, silica gel) purified, with prepare faint yellow solid -37 (15.24 milligrams, 0.0423 mM, 64.4% yield).¹H NMR (600MHz, D₂O) δ 3.44 (d, 1H, J=4.6Hz), 3.51-3.58 (m, 2H), 3.70 (dd, J= 1H, 4.6,13.2Hz), 3.75 (dd, 1H, J=7.2,11.3Hz), 3.88 (dd, 1H, J=6.2,11.3Hz), 4.10 (dd, 1H, J=3.9,8.4Hz), 4.26 (t, 1H, J=3.4,3.4Hz), 6.75 (d, 1H, J=15.8Hz), 7.58 (d, 1H, J= 15.8Hz), 7.76 (s, 4H)；¹³C NMR (150MHz, D₂O) 169.0 δ, 140.1,137.9,128.3,125.8,122.1, 74.5,71.3,60.3,59.9,59.5,41.2.HRMS calcd for [C₁₆H₁₉F₃N₂O₄+H]⁺361.1380, found 361.1370.

1.4 confirmation compounds 9 to 24

In the present embodiment, by assessment compound 9 to 24 (concentration 50uM) respectively in pH 4.6 and 7.0 (chemical chaperones Albumen can combine closely in neutral pH 7.0 with ER enzyme, and be released in acid pH 4.6 by lysosome) when to α-Gal The inhibitory activity of A.As a result it is set forth in Fig. 1.

As shown in the result of Fig. 1, in 16 kinds of compounds (i.e. compound 9 to 24) of test, only compound 17 and 18 in With the inhibitory activity for being more than 60% when pH 4.6.The discovery points out that (3R, 4S, 5R) configuration of compound 17 and 18 is in inhibition Important role is play during α-Gal A.Isomer 19 (having (2R, 3R, 4S, 5S) configuration) is when pH 4.6 Inhibit potentiality poor, then there are good inhibition potentiality in pH 7.0.Other isomers are then in vitro to α-Gal A has bad or does not have an inhibitory activity.In addition, further the inhibitory activity of analysis of compounds 17-19, table 2 elaborate those knots Fruit.

Table 2

[a]: experiment is repeated with three to measure IC₅₀Numerical value.

Inhibition potentiality (IC of the compound 17 when pH 7.0₅₀=0.053 μM) it is about its inhibition potentiality when pH 4.6 (IC₅₀=0.67 μM) 13 times.The discovery is pointed out, under simulated conditions in vitro, compound 17 has advantageous combination excellent Gesture can combine closely in ER (neutral pH) with deficiency α-Gal A enzyme, then have poor combination in lysosome (acid pH) Ability.Similarly, compound 19 in the inhibition potentiality of neutral pH be about it in 19 times of inhibition potentiality of acid pH.Relatively, Compound 18 in the inhibition potentiality of neutral environment (pH 7.0) be only about its in acidic environment (pH 4.6) inhibition potentiality 3.2 Times.Compound 17 is 264 times of compound 19 in 200 times that the potentiality of pH 7.0 are about compound 18.It is noticeable It is that the inhibitory activity of compound 18 is poor compared with the inhibitory activity of compound 17, shows the position of C2- amine methyl structural to can significant shadow Ring inhibitory activity.

1.5 confirmation compounds 33 to 37

Then assessment compound 33 to 37 (concentration 50uM) is living to the inhibition of α-Gal A when pH 4.6 and 7.0 respectively Property.Table 3 summarizes those results.

In neutral pH, in addition to compound 34, the compound 33 and 35-37 of low micromolar concentration can be to rh- α-Gal A Generation in vitro inhibits effect.In addition, all compounds all have lower inhibition potentiality when pH 4.6.Its inhibitory activity Compared with two kinds of reference compound-DGJ (IC₅₀=0.013 μM) and DIA (IC₅₀=0.75 μM) it is low.

Table 3

[a]: experiment is repeated with three to measure IC₅₀Numerical value.

[b]: reference compound is to compare analysis.

[c]: this test is that glucosides is muttered using 4MU- α-gala piperazine as substrate, when Km=0.3mM when pH 4.6, pH 7.0 Km=2.4mM.

Companion's effect of the confirmation compound 17,33 and 37 of embodiment 2

It is based on embodiment 1 as a result, selected chemicals 17,33 and 37 carry out subsequent analysis, using understand its as be mutated α- The effect of molecular chaperone protein of Gal A.It specifically, is thrown the FD disease-inflicted cells strain N215S with specific missense mutation Compound 17,33 or 37 is given, Fig. 2 is as a result set forth in.

Make us ground of being surprised, the compound 17 of various concentration (by 0 to 100 μM) can all increase the activity of mutation α-Gal A.Phase Compared with the untreated cell of control group, compound 17,33 or 37 any compounds are administered to N215S sufferer lymphocyte (100 μM) can all dramatically increase the activity (compound 17 and 33 increases separately 10 and 18 times) (the 2nd figure) of α-Gal A.In addition, changing The companion's effect for closing object 17 or 33 is all good compared with DGJ or DIA.Specifically, at same concentrations (25 μM), 33 companion's effect is about It is 4 times of DGJ, and at same concentrations (100 μM), 33 companion's effect is about 13 times of DIA.

The effect for inquiring into compound 17 and 33 pairs of other missense α-Gal A mutant respectively, it includes R112H, S148N, P205T, Q279E, R310Q, R356W and R363C, Fig. 3 summarize those results.The result of Fig. 3 is pointed out, other than S148N, Compound 17 all can produce satisfactory Chaperone Activity to those mutation, wherein compound 17 is added, (concentration is 10 or 100 μ M the activity of deficiency α-Gal A) can be increased.It also can be observed in compound 33 similar as a result, i.e. other than S148N, change It closes object 33 and can produce satisfactory Chaperone Activity in COS7 cell.

Using a series of glycosidase come the inhibition potentiality and selectivity of detection compound 17, those glycosidases include α-Portugal Polyglycoside enzyme, beta-glucosidase, α-Gal, mankind β-Gal, recombinant human acid β-glucosidase (rh-GCase) and again Group mankind's acid alpha-D-glucosidase (rh-GAA) (result is not shown), wherein DGJ is as reference compound.It can find chemical combination Object 17 is the potentiality inhibitor of human lysosomal α-Gal A, and (is originated from warp to human lysosomal GCase, rh-GAA and β-Gal The Human Lymphocytes of decomposition) etc. other glycosidases have low inhibitory activity.It is worth noting that, positive control group (DGJ) is to β- The inhibitory activity of Gal is compound 17 to 8 times of the inhibitory activity of β-Gal.Supposition is because compound 17 may be molten to cell Enzyme body β-Gal activity has poor influence power.

As for 33 inhibition potentiality and selectivity, a series of its effect to glycosidases of this experimental analysis is thermophilic comprising fat Alpha-glucosidase, the beta-glucosidase of almond, snail of hot bacillus (Bacillus stearothermophilus) Beta-Mannosidase, the alpha-Mannosidase of sword bean, the α-Gal of coffee bean, recombinant human α-Gal A and mankind's lysate β-Gal.Table 4 elaborates those results.

Table 4

Enzyme	Inhibitory activity a (%)
		Alpha-glucosidase^b	Unrestraint
Beta-glucosidase^c	Unrestraint
		Beta-Mannosidase^d	Unrestraint
Alpha-Mannosidase^e	36
		α-Gal^f	94
rh-α-Gal A^g	97
		β-Gal^h	Unrestraint

A: the concentration for testing inhibitor is 100 μM；

B: it is originated from fatty bacillus alcalophilus；

C: it is originated from almond；

D: it is originated from snail；

E: it is originated from sword bean；

F: it is originated from coffee bean；

G: recombinant human α-Gal A；

H: it is originated from mankind's lysate.

In addition to α-Gal (being originated from recombinant human α-Gal-A and coffee bean), compound 33 (100 μM) simultaneously can not be to other surveys The glycosidase of examination, which generates, inhibits effect.

In addition, even if compound 17 and 33 still will not generate toxic action (result to cell culture at concentrations up to 200 μM It does not show).

Companion's effect of 3 compound 28 and 29 of embodiment

It include compound 28 and 29 according to two kinds of analogs of 1.2 the method prepare compound 17 of embodiment；Directly survey It measures its enzymatic activity and compares the effect of those analogs and compound 17 are to rh- α-Gal using heat transfer analysis.

As a result it points out, compound 17,28 and 29 is respectively 0.053,9.5 in inhibitory activity of the pH 7.0 to rh- α-Gal A And 22.1 μM.

Heat transfer analysis is a kind of thermal degradation test relevant to fluorescence, this experiment is by measuring melting for enzyme under different condition Change temperature (Tm) to inquire into whether compound 17,28 or 29 can improve the stability of rh- α-Gal A.As a result it is set forth in table 5 respectively And the 4th figure.

Rh- α-Gal A is 51.3 DEG C in the Tm of neutral pH (7.0), is then 54.8 DEG C in the Tm of acid pH (4.6)；The knot Fruit points out that rh- α-Gal A is rather unstable in the environment of neutral pH.When compound 17 is dissolved in the enzyme solutions of neutral pH, can send out The stability of existing rh α-GalA can change with its concentration, wherein 1,10 and 100 μM of compound 17 is added can make rh- respectively 51.3 DEG C → 53.5 DEG C → 60.8 DEG C → 65.2 DEG C of transfer is presented in the Tm of α-Gal A.In comparison, compound 28 and compound 29 do not have concentration dependent influence to the stability of rh α-Gal A.

The effect of 5. compound of table, 17,28 or 29 pairs of rh α-Gal A fusion temperatures

It is interesting that the stabilization effect trend for those analogs (i.e. compound 28 and 29) observed in heat transfer analysis It is similar to the trend of its inhibitory activity.

Administering compound 17 and α-GaI A can generate the effect of adding multiplying property to embodiment 4 jointly

In the present embodiment, it will inquire into and administering chemical chaperone proteinate 17 and rh- α-Gal A work are merged to FD sufferer Replace the effect for the treatment of for enzyme.In simple terms, rh- α-Gal A is administered to the cell strain N215S for being originated from Fabry disease sufferer The compound 17 (0,1,10 and 50 μM) of (1nM) and various concentration then determines the activity of α-Gal A.As a result it is set forth in Fig. 5.

As a result it points out, compound 17 can be used as chemical chaperone albumen in itself, uses and increases intracellular lysosome-α-Gal A Activity, wherein compared to control group, the compound 17 of 50 μM of administering can increase by the work of 2 times of intracellular lysosome-α-Gal A Property (the figure B of Fig. 5).Most of all, can significantly increase when administering compound 17 (50 μM) and rh- α-Gal A (1nM) jointly Add the activity (about increasing by 9 times) (the figure A of Fig. 5) of α-Gal A entirety.Compared to independent administering rh- α-Gal A or 17, compound 17 (chemical chaperone protein for treatment) and rh- α-Gal A (ERT) can be generated significant plus be multiplied in Fabry disease cell strain N215S Effect.

Generally, formula (I) compound (especially compound 17) of the present invention is not only a kind of α-Gal A inhibitor, also It can be used as a kind of chemical chaperone albumen, use the intracellular enzyme activity for increasing the FD sufferer with mutation α-Gal A.In addition, changing Closing object 17 can administer jointly to individual in vivo with α-GalA, add multiplying property effect to generate to intracellular α-Gal A activity.

Companion's effect of 5 compound 33 of embodiment

In the present embodiment, compound 33 will be inquired into the durability of companion's effect of N215S lymphocyte.By 0-50 μM Test compound be added N215S lymphocyte cell culture fluid in, culture 4 days after, replace fresh cell culture fluid.? After removing test compound, persistently cultivate cell 0,2 or 4 day.After with PBS washing 2 times, using 4-MU- α-Gal as substrate Determine the enzymatic activity of cell homogeneous product.Fig. 6 illustrates those results.

As shown in fig. 6, activity curve can move slightly with the time.(by 0 to 4 days) during tracking, all pulses Concentration can all increase enzymatic activity significantly；The result points out that after administering 33, the enzyme of synthesis can be stable in the presence of in cell at least 4 days.When administering drug chaperone DGJ with the same terms, it is possible to find the enzymatic activity after removing DGJ, in N215S lymphocyte It still will increase, only the increased enzymatic activity can sharply decline when the 4th day.

Summarize above-mentioned, compound 33 can be used as a kind of novel drug chaperone, to handle N215S Fabry disease Other mankind α-Gal A are mutated in sufferer lymphocyte and COS7 cell.Even if the inhibitory activity to rh α-Gal A is not so good as DGJ Or DIA, it other mankind α-Gal A mutation can but produce in the lymphocyte and COS7 cell to N215S Fabry disease sufferer Raw satisfactory companion's effect.

Although disclosing specific embodiments of the present invention in embodiment above, however, it is not to limit the invention, this Those of ordinary skill in technical field that the present invention belongs to, do not depart from the principle of the present invention and spirit in the case of, when can to its into The various changes of row and modification, therefore protection scope of the present invention is when being subject to subsidiary claim institute defender.

Sequence table

<110>Academia Sinica

<120>Fabry disease is treated

<130> P2897-PCT

<150> PCT/US NO. 62/354,139

<151> 2016-06-24

<160> 8

<170> BiSSAP 1.3

<210> 1

<211> 31

<212> DNA

<213>artificial sequence

<220>

<223>forward primer

<400> 1

aggtcggatc cgacaatgca gctgaggaac c 31

<210> 2

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>reverse primer

<400> 2

ggtggaattc ttaaagtaag tcttttaatg acatctgca 39

<210> 3

<211> 38

<212> DNA

<213>artificial sequence

<220>

<223>N215S-F_ primer

<400> 3

ggccctttca aaagcccagt tatacagaaa tccgacag 38

<210> 4

<211> 38

<212> DNA

<213>artificial sequence

<220>

<223>N215S-R_ primer

<400> 4

ctgtcggatt tctgtataac tgggcttttg aaagggcc 38

<210> 5

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>Q279E-F_ primer

<400> 5

tttggcctca gctggaatga gcaagtaact cagatggcc 39

<210> 6

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>Q279E-R_ primer

<400> 6

ggccatctga gttacttgct cattccagct gaggccaaa 39

<210> 7

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>R301Q-F_ primer

<400> 7

ttcatgtcta atgacctcca acacatcagc cctcaagcc 39

<210> 8

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>R301Q-R_ primer

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ggcttgaggg ctgatgtgtt ggaggtcatt agacatgaa 39

Claims

1. the purposes of a kind of formula (I) compound, its esters, esters or solvate, to prepare a drug with cure cloth Rui Shi disease,

Wherein,

R₁It is H or with-COR₂The C optionally replaced_1-3Amine；And

R₂It is to have the alkyl or alkene that at least the naphthenic base of a substituent group or phenyl optionally replace, wherein the substituent group is choosing Group composed by free halogen, alkyl, alkylhalide group and alkoxy.

2. purposes as described in claim 1, wherein formula (I) compound can inhibit the activity of α-GAL A.

3. purposes as claimed in claim 2, wherein formula (I) compound is a human lysosomal alpha-galactosidase A (α- Galactosidase A, α-Gal A) mutant chaperone.

4. purposes as claimed in claim 3, wherein mankind's α-Gal A mutant includes a mutation, selected from by R112H, Group composed by P205T, Q279E, R301Q, R356W and R363C.

5. purposes as claimed in claim 4, wherein formula (I) compound be selected from by

AndComposed group.

6. purposes as claimed in claim 5, wherein formula (I) compound is

7. purposes as claimed in claim 5, wherein formula (I) compound is

8. purposes as claimed in claim 5, wherein formula (I) compound is

9. purposes as claimed in claim 5, wherein the drug further includes a mankind α-Gal A.

10. the compound of structure of the one kind with formula (I-1),

Wherein,

R₃It is H or-COR₂；And

11. compound as claimed in claim 11, wherein formula (I-1) compound is

12. compound as claimed in claim 11, wherein formula (I-1) compound is

13. it is a kind of to treat one suffer from or it is doubtful suffer from Fabry disease individual formula (I) compound, its esters, esters Or solvate,

Wherein,

R₁It is H or with-COR₂The C optionally replaced_1-3Amine；And

14. formula (I) compound, its esters, esters or solvate as claimed in claim 13, wherein formula (I) compound It can inhibit the activity of α-GAL A.

15. formula (I) compound, its esters, esters or solvate as claimed in claim 14, wherein formula (I) compound For the chaperone of a human lysosomal alpha-galactosidase A (α-galactosidase A, α-Gal A) mutant.

16. formula (I) compound, its esters, esters or solvate as claimed in claim 15, the wherein mankind α-Gal A Mutant includes a mutation, is selected from the group as composed by R112H, P205T, Q279E, R301Q, R356W and R363C.

17. formula (I) compound, its esters, esters or solvate as claimed in claim 16, wherein formula (I) compound Be selected from by

And Composed group.

18. formula (I) compound, its esters, esters or solvate as claimed in claim 17, wherein formula (I) compound It is

19. formula (I) compound, its esters, esters or solvate as claimed in claim 17, wherein formula (I) compound It is

20. formula (I) compound, its esters, esters or solvate as claimed in claim 17, wherein formula (I) compound It is

21. formula (I) compound, its esters, esters or solvate as claimed in claim 17, wherein being administered to the individual 0.001-500 grams formula (I) compound daily, to slow down, improve and/or prevent illness relevant to Fabry disease.

22. formula (I) compound, its esters, esters or solvate as claimed in claim 17, wherein changing in administering formula (I) Prior to, concurrently with, or after closing object, its esters, esters or solvate, the people that a therapeutically effective amount is administered to the individual is further included Class α-Gal A.

23. it is a kind of to treat one suffer from or it is doubtful suffer from Fabry disease individual method, comprising to the individual administer one Formula (I) compound, its esters, esters or the solvate of therapeutically effective amount,

Wherein,

R₁It is H or with-COR₂The C optionally replaced_1-3Amine；And

24. method as claimed in claim 23, wherein formula (I) compound can inhibit the activity of α-GAL A.

25. method as claimed in claim 24, wherein formula (I) compound is a human lysosomal alpha-galactosidase A (α- Galactosidase A, α-Gal A) mutant chaperone.

26. method as claimed in claim 25, wherein mankind's α-Gal A mutant includes a mutation, selected from by Group composed by R112H, P205T, Q279E, R301Q, R356W and R363C.

27. method as claimed in claim 26, wherein formula (I) compound be selected from by

And Composed group.

28. method as claimed in claim 27 further includes prior to, concurrently with, or after administering formula (I) compound, to the individual Administer the mankind α-Gal A of a therapeutically effective amount.

29. method as claimed in claim 28, wherein formula (I) compound is

30. method as claimed in claim 28, wherein formula (I) compound is

31. method as claimed in claim 28, wherein formula (I) compound is

32. method as claimed in claim 27, wherein being to administer daily 0.001-500 grams of formula (I) compound to the individual.

33. method as claimed in claim 32, wherein being to administer daily 0.05-450 grams of formula (I) compound to the individual.